Application of plasmalogen in preparation of medicine for promoting spinal cord injury repair

Abstract

The invention discloses application of plasmalogen in preparation of a medicine for promoting spinal cord injury repair. The invention further discloses application of the plasmalogen in preparation of drugs for regulating neuroinflammation or oxidative stress in spinal cord injury. The invention also discloses application of the plasmalogen in preparation of a medicine for promoting motor function recovery after spinal cord injury. The invention further discloses application of the plasmalogen in preparation of a medicine for neuroprotection in spinal cord injury. The invention further discloses application of the plasmalogen to preparation of a medicine for nerve cell myelin sheath regeneration after spinal cord injury. The invention also discloses application of the plasmalogen in preparation of a medicine for promoting nerve fiber regeneration after spinal cord injury. It is found that plasmalogen can effectively reduce the ROS level and regulate and control neuroinflammation and oxidative stress levels, so that neuronal axis mutability and apoptosis are reduced, and neuronal myelin regeneration is promoted.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to plasmalogens (Pls), specifically to the use of plasmalogens in preparing a medicine for promoting the repair of spinal cord injury. Background Art

[0002] Spinal cord injury (SCI) is a serious injury to the central nervous system with high disability and mortality rates. There is currently no effective clinical treatment. SCI is divided into primary and secondary injuries. Primary injury is caused by initial mechanical damage to the spinal cord. Secondary injury involves a complex process driven by multiple factors, including oxidative stress, inflammatory response, excitotoxicity, and microcirculatory disorders. Secondary injury that develops on the basis of primary injury is a continuous progressive process that plays a key role in the later recovery of spinal cord function. Therefore, effective intervention in secondary injury is crucial for injury repair.

[0003] Recent studies have shown that the increase in reactive oxygen species (ROS) and its accompanying peroxidation and severe inflammatory cascades are key factors in secondary spinal cord injury, leading to widespread neuronal apoptosis. ROS are molecules formed during the incomplete reduction of oxygen and are important mediators of inflammation. Studies have shown that excessive accumulation of ROS at the site of spinal cord injury due to the oxidation of cellular components (including proteins, lipids, and nucleic acids) leads to increased mitochondrial membrane permeability; activation of apoptotic signaling pathways; significant upregulation of apoptotic mediator Caspase-3; downregulation of anti-apoptotic protein bcl-2-associated X protein (BAX); and upregulation of pro-apoptotic protein bcl-2-associated X protein (BAX), inducing cell apoptosis. In addition, ROS exacerbate the inflammatory cascade, activate pro-inflammatory M1 macrophages / microglia, release cytotoxic factors such as IL-6, TNF-α, and IL-1β, and cause irreversible and widespread neuronal apoptosis and demyelination. Therefore, regulating the generation and elimination of ROS in the spinal cord injury area to achieve internal balance is a potential therapy for treating inflammation and reducing neuronal apoptosis.

[0004] Lipids have multiple roles, the most important of which is to ensure the normal structure and function of biological membranes and play an important messenger role in maintaining the homeostasis of ROS. Plasmalogens (Pls) are a special subgroup of phospholipids and one of the main components of biological membranes. They are widely found in marine invertebrates, especially in sea squirts, mussels and sea cucumbers. More than 50% of Pls are found in neurons and synapses. In humans, plasmalogens (Pls) are also abundant, with particularly high expression in the brain and heart. Plasmalogens (Pls) were originally widely regarded as cellular antioxidants containing vinyl ether-linked fatty alcohols at the sn-1 position of the glycerol backbone and ester-linked fatty acids at the sn-2 position, which can bind to ROS and thus reduce the accumulation of ROS in tissues. For various nerve cells in the nervous system, plasmalogens (Pls) have been found to reduce microglial activation and neuroinflammation; plasmalogens Pls can also reduce neuronal apoptosis and promote dendritic spine elongation, thereby improving learning and memory. Studies have shown that reduced levels of plasmalogens (Pls) in the cortex and hippocampus of patients with Alzheimer's disease are closely associated with the severity of dementia. Scallop-derived plasmalogens (Pls) are currently being used in preclinical and clinical studies and have been shown to improve memory function in patients with mild AD to prevent and / or slow disease progression.

[0005] Although there is convincing evidence that plasmalogens (Pls) have a protective effect on neurological diseases, no studies have reported the therapeutic effects of plasmalogens (Pls) on spinal cord injury or the potential mechanisms involved. In the present invention, we explored the protective effects of plasmalogens (Pls) on neuronal axonal degeneration and apoptosis after spinal cord injury. The present invention aims to determine whether exogenously supplemented plasmalogens (Pls) from scallops and sea cucumbers can promote neural regeneration and functional repair in a microenvironment rich in ROS. Summary of the invention

[0006] In view of the above technical problems in the prior art, the present invention provides the use of plasmalogen in the preparation of a drug for promoting the repair of spinal cord injury, and the use is intended to solve the technical problem that the drugs in the prior art are not effective in treating spinal cord injury.

[0007] The present invention provides the use of plasmalogen in preparing a medicine for promoting the repair of spinal cord injury.

[0008] Specifically, promoting the repair of spinal cord injury is achieved through the regulation of neuroinflammation and oxidative stress. The regulation of neuroinflammation and oxidative stress can promote the repair of spinal cord injury and inhibit the inflammatory response.

[0009] The present invention also provides the use of plasmalogen (Pls) in preparing a drug for regulating neuroinflammation or oxidative stress in spinal cord injury.

[0010] The present invention also provides the use of plasmalogen (Pls) in preparing a medicine for promoting recovery of motor function after spinal cord injury.

[0011] Specifically, the recovery of motor function is manifested as improved motor ability.

[0012] The present invention also provides the use of plasmalogen (Pls) in preparing a drug for neuroprotection in spinal cord injury.

[0013] The present invention also provides the use of plasmalogen (Pls) in preparing a medicine for regenerating neuronal myelin after spinal cord injury.

[0014] The present invention also provides the use of plasmalogen (Pls) in preparing a medicine for promoting the regeneration of nerve fibers after spinal cord injury.

[0015] Specifically, the structural formula of plasmalogen is shown below,

[0016]

[0017] The present invention detects the expression of neuronal myelin protein MBP and the axon elongation of DRG cell model neurons (TUJ-1) by immunofluorescence and western blotting techniques, and then verifies that plasmalogen (Pls) is achieved by reducing neuronal axonal damage and promoting neuronal remyelination. By detecting ROS levels, MDA oxidative stress levels and immunofluorescence detection of the expression levels of M1 pro-inflammatory protein iNOS and M2 anti-inflammatory protein ARG-1, the changes in neuroinflammation and oxidative stress in cells and spinal cord tissues of each experimental group are compared, and it is found that plasmalogen (Pls) can effectively reduce ROS levels and alleviate inflammation.

[0018] The present invention discloses the application of plasmalogens (Pls) in promoting the repair of spinal cord injury. The present invention evaluates the repair effect and the intrinsic mechanism of action by multiple methods such as immunofluorescence staining, oxidative stress level detection, protein blotting technology, behavioral scoring, etc., establishes a cell model under the stimulation of highly reactive oxygen species (ROS) in vitro and an animal model of complete spinal cord transection in mice, and finds that plasmalogens (Pls) can effectively reduce ROS levels, regulate neuroinflammation and oxidative stress levels, thereby reducing neuronal axonal degeneration and apoptosis, and promoting neuronal myelin regeneration. Therefore, plasmalogens (Pls) can promote the recovery of motor function of spinal cord injury, and provide a promising treatment method for clinical SCI repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The establishment and detection of high ROS cell model in mouse dorsal root ganglion DRG Figure 1 A is the immunofluorescence staining of TUJ-1 in neuronal axons stimulated by the strong oxidant H2O2; Figure 1 B is a statistical graph of the length of TUJ-1 neuron axons; Figure 1 C is the detection of intracellular ROS production before and after plasmalogen (Pls) treatment; Figure 1 D is a statistical graph of cellular ROS level detection.

[0020] Figure 2 The cell model of macrophage RAW 264.7 under high ROS was established and the inflammatory level was detected; Figure 2 A is the detection and fluorescence intensity analysis of ROS in cells before and after plasmalogen (Pls) intervention; Figure 2 B is the immunofluorescence image of the M1 pro-inflammatory (iNOS) phenotype and the M2 anti-inflammatory (ARG-1) phenotype of cells before and after plasmalogen (Pls) intervention, as well as the corresponding fluorescence intensity analysis graph.

[0021] Figure 3 The oxidative stress and inflammation levels were detected in mice 7 days after spinal cord injury. Figure 3 A is the fluorescence detection of ROS level in damaged tissue; Figure 3 B is the statistical graph of ROS level and MDA detection; Figure 3 C is the results and statistical graph of Western blotting to detect the levels of anti-inflammatory protein INOS and endogenous antioxidant enzyme SOD2; Figure 3 D is the tissue immunofluorescence image of M1 pro-inflammatory (iNOS) phenotype and M2 anti-inflammatory (ARG-1) phenotype.

[0022] Figure 4 This is a study on neural stem cell injury and myelin regeneration after spinal cord injury in mice. Figure 4 A is a tissue immunofluorescence image of apoptotic protein Caspase-3 and neuronal apoptosis TUNEL / TUJ-1, as well as a tissue immunofluorescence image of nestin / astrocytic GFAP 7 days after spinal cord injury, which evaluates neuronal apoptosis and quantifies the accumulation of nestin-positive neural stem cells with differentiation potential in the center of spinal cord injury; Figure 4 B is the level of TUJ-1 protein in the injured central neurons detected by Western blotting 7 days after injury and its statistical graph, Figure 4 C-4D is the tissue immunofluorescence image and Western blot detection and statistical graph of myelin protein MBP 6 weeks after injury.

[0023] Figure 5 It is a behavioral and histological test after spinal cord injury in mice to evaluate the effect of neurological recovery. Figure 5 A is Nissl staining; Figure 5 B is the BMS score used to assess the hind limb activity level of mice at each time point after injury; Figure 5C is the tissue immunofluorescence staining of CS-56 in glial scar and MAP2 in mature neurons 6 weeks after injury.

[0024] Figure 6 This is a schematic diagram of the principle of the present invention, which briefly describes that this study is to promote nerve repair and protection by regulating the immune microenvironment of oxidative stress and neuroinflammation through exogenous administration of acetylphospholipids (Pls) after spinal cord injury. This effect is exerted by regulating the polarization state of microglia, reducing neuronal axon degeneration and apoptosis, and promoting myelin regeneration. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] 1. Experimental Materials

[0028] 1. Experimental Animals

[0029] The experimental animals were adult female C57BL / 6N mice, 6 weeks old, weighing 17-20 g, purchased from Spefoc (Suzhou) Biotechnology Co., Ltd. (Experimental Animal License No.: SCXK (Suzhou) 2022-0006, Experimental Animal Certificate No.: NO.202377188). The experimental animals were housed in the standard animal room of Jiangnan University, with a room temperature of 23±2°C, a relative humidity of 55%±5%, a light-dark cycle of 12 h (6:00-18:00), a quiet environment, and free access to food and water. This experimental study was approved by the Ethics Review Committee of Jiangnan University (JN.No20230615c0720905

[312] ).

[0030] 2. Experimental drugs

[0031] Antibodies: Tuj-1 (abclonal, Catalog No.: A17074), MAP2 (Proteintech, Catalog No.: 17490-1-AP), GFAP (Proteintech, Catalog No.: 60190-1-Ig), MBP (Proteintech, Catalog No.: 10458-1-AP), iNOS (Proteintech, Catalog No.: 18985-1-AP), ARG-1 (Proteintech, Catalog No.: 66129-1-AP), CS-56 (Mi lliporeSigma, catalog number: C8035), Caspase-3 (Proteintech, catalog number: 10380-1-AP), Nestin (Boster, catalog number: BA1289), SOD2 (Proteintech, catalog number: 24127-1-AP), Actin (abclonal, catalog number: ARC5115-01), horseradish peroxidase-labeled goat anti-rabbit IgG (Boster), horseradish peroxidase-labeled goat anti-mouse IgG (Boster).

[0032] Reactive oxygen species detection kit (Biyuntian, catalog number: S0033S), malondialdehyde (MDA) assay kit (TBA method) (Nanjing Jiancheng Bioengineering Institute, A003-2), TUNEL BrightRed Apoptosis Detection Kit (Nanjing Novogene Biotechnology Co., Ltd.), DAPI (Thermo Fisher), Nissl staining solution (toluidine blue method) (Beijing Solebao Technology Co., Ltd.)

[0033] Lipopolysaccharide LPS (sigma), plasmalogen (Larodan, Catalog No.: 39-0225),

[0034] The structural formula of plasmalogen is shown below.

[0035]

[0036] Mouse mononuclear macrophage RAW 264.7 (Wuhan PriCells Biotechnology Co., Ltd.).

[0037] 3. Main reagents and instruments

[0038] Carbon dioxide incubator (Thermo Fisher Scientific (China) Co., Ltd., model: 371GP), biological safety cabinet (Shanghai Lishen Scientific Instrument Co., Ltd., model: HFsafe-1500LC (A2)), small animal anesthesia machine (Shenzhen Ruiwode Life Science Technology Co., Ltd.), upright fluorescence microscope (Carl Zeiss, Germany, model: Axio Imager Z2), fully automatic frozen section machine (Leica Pathology System, Germany, model: CM3050S), low-temperature ultracentrifuge (Thermo Fisher, USA, model: thermo / Micro21R), low-temperature frozen tissue grinder (Shanghai Jingxin Industrial Development Co., Ltd., model: TISS-48), developer (Shanghai Tianneng Technology Co., Ltd., model: Tanon-5200Multi), spectrophotometer (Boten Instrument Co., Ltd., USA, model: Epoch), digital slice scanner (3DHISTECH, Hungary, model: PANNORMICMIDI), stereo microscope (supplied by Jiangnan Yongxin, model: JSZ6S).

[0039] 2. Experimental Methods

[0040] 1. Cell-related experiments

[0041] Extraction and culture of mouse primary cells

[0042] Newborn C57BL / 6 mice were euthanized within 12 hours of birth after disinfection with 75% alcohol by volume for 5 minutes. Under a stereoscope, the back skin was cut open, the spinal canal was opened, and the spinal cavity was exposed. The dorsal root ganglion cells between the vertebral foramina were removed with microvascular forceps, temporarily stored in pre-cooled high-glucose DMEM, and their membranes were carefully peeled off and then placed in wells pre-coated with poly-lysine (Sigma, USA). The cells were placed in DMEM supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA) by volume, incubated at 37°C for 12 hours, and then transferred to complete culture medium for experiments. The complete culture medium consisted of DMEM / F12 (Gibco, USA), 1% nonessential amino acids (Gibco, USA), 1% sodium pyruvate (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA), and 2% B27 supplement (Gibco, USA).

[0043] 2. Animal-related experiments

[0044] (1) Establishment and care of the mouse spinal cord complete transection injury model

[0045] The gas flow rate of the small animal anesthesia machine was set to 2L / min, and the mice were connected to a breathing mask to induce anesthesia. After the mice entered a deep anesthesia state, the gas flow rate was set to 0.5L per minute to maintain anesthesia. The mice were fixed on the operating table, the skin was prepared and disinfected, a 1cm incision was made at the T8-T9 segment of the spine, the paravertebral muscles were bluntly separated, the T8-T9 lamina was exposed and removed, the surgical suture was passed from the ventral side of the spinal cord to the contralateral side, the spinal cord was slightly lifted, and the T8-T9 segment spinal cord was completely transected with microscissors to ensure that the transection was clean and accurate to avoid damage to the surrounding nerve tissue. Immediately after surgery, 2μl PBS or plasmalogen (Pls) (1mg / ml) was injected into the transected gap. After these treatments, the skin and muscle tissues were sutured, and the mice were returned to the cage for recovery. Penicillin was injected intramuscularly for 3 consecutive days after surgery to prevent infection, and 100ul PBS or plasmalogen (Pls) 4ug / mouse was intraperitoneally administered. The mouse bladder was massaged twice a day to assist urination, and the bedding was changed frequently to keep the cage dry and clean. The postoperative conditions of the mice were closely monitored, including respiration, heart rate, and eating and drinking.

[0046] BMS score

[0047] After the experimental mice underwent complete spinal cord transection surgery, their motor function was evaluated in weeks, and the evaluation standard was BMS (Basso Mouse Scale). The evaluation process was carried out in a spacious and bright environment to ensure that the mice had enough space for activities. The scoring details were as follows, and two people performed double-blind scoring. The BMS scoring standard is detailed in Table 1.

[0048] After the experimental mice underwent complete spinal cord transection surgery, their motor function was evaluated in weeks, and the evaluation standard was BMS (Basso Mouse Scale). The evaluation process was carried out in a spacious and bright environment to ensure that the mice had enough space for activities. The scoring details were as follows, and two people performed double-blind scoring. The BMS scoring standard is detailed in Table 1.

[0049] Table 1

[0050]

[0051]

[0052] (3) Spinal cord tissue sampling and frozen sections

[0053] On the 7th day and 6th week after injury, 3 mice were taken from each group for cardiac perfusion sampling. A sodium pentobarbital solution with a concentration of 5 mg / ml was prepared as an anesthetic. After filtering with a 0.22um bacterial filter, the mice were anesthetized at a dose of 50 mg / kg. After the mice entered the anesthetized state, they were fixed on the sampling board, the chest and abdominal cavity of the mice were opened and the heart was fully exposed. The perfusion needle was inserted into the appropriate depth along the apex direction, the left atrial appendage was cut open, and 4°C pre-cooled saline was first injected with a syringe. After the liver color turned from red to white, 4% paraformaldehyde was used for cardiac perfusion for internal fixation. With the spinal cord injury as the center, spinal cord tissue with a length of about 3 cm was retained and fixed in 4% paraformaldehyde. After gradient dehydration with sucrose of 20% and 30% by mass percentage, the spinal cord tissue was embedded with OCT after it could settle to the bottom in the 30% sucrose solution, and the block was stored in -80°C freezer. The embedded frozen spinal cord tissue was then sliced ​​longitudinally and continuously from dorsal to ventral with a thickness of 14 μm, marked with slice names and grouped, and stored frozen at -80°C.

[0054] 3. Experimental Results

[0055] 1. Conduct cell experiments to verify that acetylphospholipid Pls can effectively reduce neuronal axon degeneration and cell apoptosis.

[0056] The specific steps are as follows:

[0057] ① Extract primary DRG cells as described above, randomly divide them into two groups, and then conduct the following experiment.

[0058] Control group: cells were treated with 400 μM H2O2 and incubated in a cell culture incubator for 24 h.

[0059] Experimental group: cells were treated with 400 μM H2O2 + 400 ng / ml plasmalogen (Pls) and incubated in a cell culture incubator for 24 hours.

[0060] ②After completing step ①, perform TUJ-1 immunofluorescence staining

[0061] See Figure 1 A, Compared with the control group, the axonal degeneration of neurons in the plasmalogen (Pls) group was significantly reduced. The axonal length of DRG neurons was quantified by ImageJ analysis ( Figure 1 B), Results showed that plasmalogen (Pls) significantly increased axon length.

[0062] ③After completing step ①, the ROS level was detected using a ROS probe. The results showed that the ROS level was significantly reduced after plasmalogen (Pls) treatment ( Figure 1 C and 1D).

[0063] These results suggest that plasmalogens (Pls) can effectively reduce H2O2-induced neuronal axonal degeneration.

[0064] 2. Verify that plasmalogen Pls reduces ROS levels and regulates macrophage / microglia phenotypes. Perform cell experiments. The specific steps are as follows:

[0065] ① Experimental groups: control group, LPS stimulation group, LPS+Pls group.

[0066] Control group: RAW 264.7 cells were stimulated with PBS blank control and incubated in a cell culture incubator for 24 hours.

[0067] LPS stimulation group: RAW 264.7 cells were stimulated with 1 mg / ml LPS and incubated in a cell culture incubator for 24 hours.

[0068] LPS+Pls group: RAW 264.7 cells were stimulated with 1 mg / ml LPS+1 μg / ml Pls and incubated in a cell culture incubator for 24 hours.

[0069] ②After completing step ①, stain with ROS probe

[0070] After 24 h of treatment with 1 μg / ml Pls, cells showed a decrease in ROS levels ( Figure 2 A)

[0071] ③After completing step ①, use immunofluorescence staining technology to detect protein expression

[0072] The results showed that the expression of iNOS decreased and ARG-1 increased in the experimental group with 1 μg / ml Pls. Figure 2 B). The proportion of iNOS positive cells decreased from 73.37% to 46.53%, and the proportion of ARG-1 positive cells increased from 19.31% to 66.81%.

[0073] Macrophages / microglia with increased ARG-1 expression tend to inhibit inflammation, induce angiogenesis, and promote tissue repair, thereby exhibiting an anti-inflammatory M1 phenotype, while macrophages / microglia with increased iNOS expression have the opposite effect, promoting inflammation and neurotoxicity. The results showed that LPS+Pls-treated macrophages / microglia had reduced ROS generation and increased the pro-inflammatory M1 / anti-inflammatory M2 ratio, which helped to reduce inflammation.

[0074] 3. To verify that plasmalogen Pls has a promoting effect on the repair of spinal cord injury, animal experiments were conducted. The specific steps are as follows:

[0075] ① Animal experiment: The mice were randomly divided into two groups, with 8 mice in each group, and the following experiments were performed:

[0076] SCI group: simple spinal cord transection injury, 2ulPBS was added to the injury center during the operation.

[0077] Pls group: spinal cord complete transection injury, 2ul of plasmalogen (Pls) (1mg / ml) was added to the injury center during surgery.

[0078] ②After completing step ①, take tissue samples 7 days after surgery

[0079] ③After completing step ①, take tissue samples 6 weeks after surgery

[0080] ④Complete step ② to detect the levels of ROS and malondialdehyde (MDA).

[0081] In the plasmalogen (Pls)-treated group, ROS levels were significantly decreased ( Figure 3 A), and the MDA level associated with ROS damage was also significantly reduced ( Figure 3 B).

[0082] ⑤After completing step ②, tissue samples were taken 7 days after surgery for Western blotting to detect the expression of superoxide dismutase (SOD2) and iNOS, and immunofluorescence to detect the expression of ARG-1 and INOS.

[0083] Western blotting results showed that superoxide dismutase (SOD2) is an enzyme that catalyzes the production of superoxide free radicals (O 2- ) is decomposed into H2O2 and O2, and its expression increases in the local tissue of the injured spinal cord; after Pls treatment, the expression of the pro-inflammatory protein iNOS decreases ( Figure 3 C).

[0084] The same results were observed by immunofluorescence, with decreased expression of pro-inflammatory iNOS and increased expression of anti-inflammatory marker ARG-1 ( Figure 3 D).

[0085] After spinal cord injury, increased local ROS generation leads to an inhibitory microenvironment characterized by high levels of oxidative stress and inflammation. The above results show that Pls effectively regulates the effects of oxidative stress and inflammation in mice after spinal cord injury, creating a suitable neural regeneration environment for mice, thereby playing a neuroprotective role.

[0086] It was found that plasmalogens (Pls) can reduce neuronal apoptosis after spinal cord injury and promote axon regeneration and myelination.

[0087] ⑥After completing step ②, take frozen sections of the tissue samples 7 days later and perform immunofluorescence staining of TUNEL / TUJ-1, Caspase-3, and Nestin.

[0088] The results showed that after treatment with plasmalogen Pls, the expression of Caspase-3, a neuronal apoptosis marker, was reduced, and the positive rate of TUNEL staining was reduced ( Figure 4 A), the number of newly born neurons TUJ-1 increased ( Figure 4 B). At the same time, we observed an increase in the expression of Nestin-positive cells with differentiation potential in the injured center of the spinal cord, indicating that after plasmalogen (Pls) treatment, the number of neural stem cells with neural differentiation potential increased, which may be due to the migration or activation of endogenous neural stem cells, ultimately promoting the differentiation and regeneration of neurons.

[0089] ⑦ After completing step ③, take frozen sections of the 6-week tissue samples for MBP immunofluorescence staining, and take fresh proteins of the 6-week tissue samples for Western blotting to detect the expression of MBP.

[0090] After spinal cord injury, axonal degeneration impairs neural signaling, and myelin plays a key role in maintaining axonal integrity. The expression of the myelin protein MBP was evaluated in the late stage of spinal cord recovery (6 weeks). Immunofluorescence staining and Western blotting techniques showed increased MBP levels, indicating increased neuronal myelination ( Figure 4 C and 4D).

[0091] The above results show that plasmalogen (Pls) treatment can effectively reduce early neuronal apoptosis after spinal cord injury, increase the survival of endogenous neural stem cells, and promote the regeneration of neurons. In addition, in the later stage of recovery, it was found that this effect helps the regeneration of neuronal axons and remyelination.

[0092] ⑧After completing step ③, take 6-week tissue samples for Nissl staining and immunofluorescence staining.

[0093] Nissl staining showed that the number of Nissl-positive neurons in the injured area of ​​mice treated with plasmalogen (Pls) increased ( Figure 5 A). Immunofluorescence staining showed that the proportion of mature neuron marker MAP2 in the center of the injured spinal cord increased significantly and was arranged in a linear and orderly manner ( Figure 5 C). In addition, CS-56 staining, used to assess scar formation during the recovery phase, showed that the size of CS-56-positive areas in the injured spinal cord of plasmalogen (Pls)-treated mice was significantly reduced.

[0094] ⑨After completing step ①, perform BMS scoring on mice every week.

[0095] BMS scores showed that the plasmalogen (Pls) group significantly promoted the recovery of hind limb motor function in mice ( Figure 5 B).

[0096] The above results show that acetalphospholipid Pls treatment alleviated spinal cord tissue damage, protected neurons in the injured area, and promoted the recovery of motor function in mice with spinal cord injury.

[0097] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of plasmalogen in the preparation of drugs for promoting the repair of spinal cord injury.

2. Use of plasmalogens in the preparation of drugs for regulating neuroinflammation or oxidative stress in spinal cord injury.

3. Use of plasmalogen in the preparation of a drug for promoting recovery of motor function after spinal cord injury.

4. Use of plasmalogen in the preparation of drugs for neuroprotection in spinal cord injury.

5. Use of plasmalogen in the preparation of a drug for neuronal myelin regeneration after spinal cord injury.

6. Use of plasmalogen in the preparation of drugs for promoting nerve fiber regeneration after spinal cord injury.

7. The use according to claims 1-6, characterized in that The structural formula of the plasmalogen is shown below,

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