Application of N-acetylminosamine in drug preparation

Abstract

This invention relates to the application of N-acetylmnosamine in pharmaceutical preparation, belonging to the field of biomedical technology. This invention provides the application of N-acetylmnosamine in the preparation of drugs for treating or preventing neurological dysfunction caused by hydrocephalus; in the preparation of drugs for treating or preventing motor disorders caused by hydrocephalus or neurodegenerative diseases; in the preparation of drugs for improving cognitive impairment; in the preparation of drugs for inhibiting astrocyte proliferation and promoting astrocyte transformation; and in the preparation of drugs for reducing white matter demyelination damage in brain tissue. Through the application of ManNAc provided by this invention, new drugs will be provided for the prevention and treatment of neurological dysfunction caused by hydrocephalus, offering new treatment directions for patients.

Description

Technical Field

[0001] This invention relates to the application of N-acetylmnosamine in the preparation of drugs, belonging to the field of biomedical technology. Background Technology

[0002] Hydrocephalus refers to the dilation of the ventricles caused by impaired production, circulation, or absorption of cerebrospinal fluid. Based on different etiologies, hydrocephalus can be divided into primary and secondary types. Primary hydrocephalus is caused by congenital or hereditary abnormalities in cerebrospinal fluid circulation or absorption, such as congenital ventricular stenosis or post-meningitis syndrome. Secondary hydrocephalus is caused by other diseases or trauma resulting in impaired cerebrospinal fluid circulation or absorption, such as brain tumors, cerebral hemorrhage, or brain injury. In addition, neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), can also lead to atrophy of the cerebral cortex, subsequently causing ventricular dilation.

[0003] Hydrocephalus, including ventricular enlargement secondary to neurodegenerative diseases, can cause periventricular white matter damage, abnormal proliferation and polarization of glial cells, leading to a series of neurological dysfunctions, including motor disorders, cognitive impairments, and visual impairments. Motor disorders, in particular, have received significant attention from the academic community. Muscle weakness, decreased balance and coordination, and involuntary muscle movements severely impact patients' daily lives and can lead to falls and other life-threatening risks.

[0004] In recent years, hydrocephalus has become a serious public health problem, occupying an increasingly important position in the spectrum of human diseases. Statistics show that idiopathic normal pressure hydrocephalus (a common type of hydrocephalus in the elderly) accounts for 0.5%-1.5% of the population aged 60 and above in my country. Patients with other causes are approximately 2-3 times more likely to have idiopathic normal pressure hydrocephalus.

[0005] However, the treatment of hydrocephalus currently faces significant challenges, lacking effective treatments to slow disease progression. Besides removing the primary cause, cerebrospinal fluid shunt surgery is the first-line treatment for hydrocephalus, with specific procedures including ventriculoperitoneal drainage, ventriculoatrial drainage, and lumbar cistern-peritoneal drainage. However, surgical treatment of hydrocephalus remains controversial. First, the long-term efficacy of surgery is questionable; long-term follow-up studies show that factors such as drainage duct occlusion can lead to a gradual decline in patient efficacy over time, with approximately 30-50% of patients requiring repeat surgery. Second, surgery is invasive, with a high incidence of complications such as subdural hematoma and infection, which some elderly patients with serious underlying diseases cannot tolerate. Therefore, exploring pharmacological intervention strategies for hydrocephalus is imperative.

[0006] Neu5Ac is a natural carbohydrate compound with a nine-carbon sugar backbone and is one of the most common sialic acids in the human body. The free form of Neu5Ac is scarce, and it is primarily involved in the formation of terminal glycan chains in cellular glycoproteins and glycolipids, playing a crucial role in mediating cell recognition, adhesion, and migration. In the nervous system, Neu5Ac content is high, estimated to be about 20 times higher than the average content in other tissue systems. In brain tissue, Neu5Ac mainly participates in the formation of gangliosides and glycoproteins, playing a unique and critical role in synapse formation, nerve impulse transmission, and maintaining neuronal and myelin homeostasis. Studies have shown that a deficiency of Neu5Ac and its complexes can lead to a series of clinical symptoms similar to hydrocephalus, such as decreased motor function and cognitive impairment. Exogenous supplementation of N-acetyl-D-mannosamine (ManNAc), a precursor in the Neu5Ac synthesis pathway, can partially reverse these clinical symptoms. Therefore, ManNAc holds promise for playing an important role in the prevention or treatment of motor disorders in hydrocephalus. ManNAc, a precursor for the synthesis of Neu5Ac molecules, is an uncharged monosaccharide with a molecular weight of 221 Daltons. ManNAc has shown potential efficacy in the treatment of motor disorders caused by some neuromuscular diseases. For example, GNE myopathy is an autosomal recessive genetic disorder caused by mutations in the GNE gene, which encodes a crucial rate-limiting enzyme in Neu5Ac synthesis. Due to the deficiency of the GNE enzyme, the Neu5Ac content in the muscle tissue of patients is significantly reduced, leading to insufficient sialylation of glycans and consequently, symptoms such as muscle weakness. Animal and clinical trials have shown that ManNAc supplementation can effectively increase sialylation of glycans in muscle cells and improve motor disorders. Furthermore, ManNAc also has a certain protective effect against cognitive impairment, potentially delaying cognitive decline in middle-aged and elderly individuals by improving spatial memory, working memory, and abnormal sleep rhythms. However, ManNAc has not yet been effectively used in the treatment of motor disorders caused by hydrocephalus, including those associated with neurodegenerative diseases. Summary of the Invention

[0007] Given the current lack of effective treatments for neurological dysfunction, especially motor dysfunction, caused by hydrocephalus, the purpose of this invention is to solve the technical problem of the application of ManNAc in the preparation of medicaments for the treatment or prevention of neurological dysfunction caused by hydrocephalus.

[0008] To achieve the objectives of this invention, the present invention provides the use of N-acetylmnosamine in the preparation of medicaments for treating or preventing neurological dysfunction caused by hydrocephalus.

[0009] This invention provides the use of N-acetylmnosamine in the preparation of medicaments for the treatment or prevention of motor disorders caused by hydrocephalus or neurodegenerative diseases.

[0010] This invention provides the use of N-acetylmnosamine in the preparation of drugs to improve cognitive impairment.

[0011] This invention provides the use of N-acetylmnosamine in the preparation of drugs that inhibit astrocyte proliferation and promote astrocyte transformation.

[0012] This invention provides the use of N-acetylmnosamine in the preparation of drugs that alleviate white matter demyelination damage in brain tissue.

[0013] Preferably, the dosage form of the drug in the above applications includes tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] ManNAc is an important precursor in the endogenous Neu5Ac biosynthesis pathway, enabling efficient intracellular synthesis of Neu5Ac. Its molecule is neutral and easily absorbed by cells. Furthermore, literature reports that compared to direct Neu5Ac supplementation, ManNAc supplementation maintains effective blood concentrations for a longer period in animal experiments and can effectively increase the concentration of Neu5Ac in the brain. All of these factors contribute to its potential application as a treatment for neurological dysfunction caused by hydrocephalus.

[0016] From a safety perspective, multiple clinical trials of ManNAc for the treatment of neuromuscular diseases and glomerulonephritis have shown no serious adverse reactions at oral doses, with only a few subjects reporting mild gastrointestinal adverse reactions. The drug has a good safety profile.

[0017] From an efficacy perspective, this invention, through the construction of an animal model of hydrocephalus, showed that after using ManNAc on the animal model, abnormal activation and polarization of astrocytes and white matter demyelination damage in the model's brain tissue pathology were inhibited; periventricular high signal intensity on magnetic resonance imaging was reduced; and motor impairment and long-term cognitive impairment in the model were significantly improved. Therefore, this demonstrates that ManNAc has a medicinal effect in treating neurological dysfunction caused by hydrocephalus.

[0018] This invention provides the application of ManNAc in the preparation of drugs for treating or preventing neurological dysfunction caused by hydrocephalus. This drug has a small molecular weight, can cross the blood-brain barrier, is easily absorbed by cells, maintains effective blood concentrations for a long time, and has few side effects on the human body, showing promising clinical application prospects. If this invention can be applied clinically, it will become a novel drug for the prevention and treatment of neurological dysfunction caused by hydrocephalus, providing patients with a new treatment direction. Attached Figure Description

[0019] Figure 1Figure showing the results of a concentration gradient experiment of ManNAc in a mouse model of hydrocephalus;

[0020] Figure 2 Figure showing the effect of ManNAc on astrocyte proliferation and polarization in a mouse model of hydrocephalus;

[0021] Figure 3 Figure showing the effect of ManNAc on periventricular white matter demyelination damage in a mouse model of hydrocephalus.

[0022] Figure 4 The effect of ManNAc on periventricular hyperintensity on MRI in a mouse model of hydrocephalus;

[0023] Figure 5 Figure showing the effect of ManNAc on motor dysfunction in a mouse model of hydrocephalus;

[0024] Figure 6 Figure showing the effect of ManNAc on long-term cognitive impairment in a hydrocephalus model mouse. Detailed Implementation

[0025] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings:

[0026] This invention provides the use of N-acetylmnosamine in the preparation of medicaments for treating or preventing neurological dysfunction caused by hydrocephalus.

[0027] This invention provides the use of N-acetylmnosamine in the preparation of medicaments for the treatment or prevention of motor disorders caused by hydrocephalus or neurodegenerative diseases.

[0028] This invention provides the use of N-acetylmnosamine in the preparation of drugs to improve cognitive impairment.

[0029] This invention provides the use of N-acetylmnosamine in the preparation of drugs that inhibit astrocyte proliferation and promote astrocyte transformation.

[0030] This invention provides the use of N-acetylmnosamine in the preparation of drugs that alleviate white matter demyelination damage in brain tissue.

[0031] The dosage forms of the drugs mentioned in the above applications include tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

[0032] The molecular chemical structure of ManNAc is as follows:

[0033]

[0034] Hydrocephalus and ventricular enlargement induced by neurodegenerative diseases can lead to focal or widespread cerebral hypoperfusion, resulting in cellular ischemia and hypoxia. This reduces the synthesis of endogenous Neu5Ac and its complexes, leading to insufficient sialylation of various sugar chains in tissues and causing damage to normal neurological functions, including motor function. Therefore, supplementing with substances related to Neu5Ac synthesis, especially easily absorbed ManNAc, plays an important biological role in maintaining nervous system homeostasis and alleviating neurological dysfunction caused by hydrocephalus.

[0035] Example

[0036] 1. Preparation of animal models and administration of drugs:

[0037] A mouse model of hydrocephalus was established by injecting kaolin into the cisterna magna (the control group received saline). After modeling, the model group received a subcutaneous injection of 0.3 ml of ManNAc solution at appropriate concentrations (ManNAc dosage gradient: 0.25, 0.5, 1 g / kg or 0.3 ml saline); the control group received a subcutaneous injection of 0.3 ml saline. Administration was repeated twice daily at 12-hour intervals for 14 consecutive days. To determine the optimal dose of ManNAc, a staggered walking test and GFAP immunohistochemistry were performed on the hydrocephalus model mice on day 7. These two indicators reflect the motor coordination ability and pathological changes in astrocytes in the hydrocephalus model mice. Results are as follows: Figure 1 As shown, compared with the model group, only the 0.5 g / kg ManNAc treatment group significantly reduced both the misstep rate and the area of ​​GFAP-positive regions. Therefore, 0.5 g / kg was used as the dosage concentration of ManNAc.

[0038] Detailed experimental procedure:

[0039] 1.1 Animal Model Preparation: Adult male C57BL6 mice weighing 27-30 grams were selected. After induction anesthesia with 1%-2% isoflurane, the mice were fixed on a stereotaxic apparatus. The angle of the mouth or ear rod was adjusted to tilt the head to form a 120° angle with the body. The anesthesia machine was turned on and continuous anesthesia with 1.5% isoflurane was administered. The skin of the cisterna magna was prepared and disinfected with povidone-iodine. The subcutaneous soft tissue was dissected with sterile ophthalmic scissors to expose the cisterna magna. The dura mater covering the cisterna magna was wiped with alcohol swabs. Then, a microsyringe with a 30-gauge needle was bent at 30-45° and 10 μL of 10% kaolin suspension (100 mg / ml dissolved in 0.9% saline) was drawn into the cisterna magna through the dura mater. The needle was inserted slowly 1.5-2.0 mm into the cisterna magna and the 10 μL of kaolin suspension was slowly injected into the basal cisternae (approximately 1 μL / min). After leaving the needle in place for 5 minutes, slowly withdraw the needle. Observe for any backflow, then suture the surgical incision and disinfect. Place the surgical animal in a 37°C electric incubator for resuscitation. Once the animal has recovered, return it to the rearing platform. Animals in the sham surgery group were injected with 10 μL of sterile saline using the same method.

[0040] 1.2 Drug Administration: Immediately after modeling, drugs were administered according to the groups, twice daily at 12-hour intervals. The control and model groups received 0.3 ml of physiological saline per animal subcutaneously each time. The ManNAc treatment group received two doses, each time a subcutaneous administration of ManNAc dissolved in 0.3 ml of physiological saline, at doses of 0.25, 0.5, and 1 g / kg / animal, respectively. All three groups were administered drugs continuously for 14 days.

[0041] 1.3 Frozen Sections of Brain Tissue: After drug administration on day 7, experimental animals were perfused and fixed. Anesthetized with pentobarbital via intraperitoneal injection, the thoracic cavity was opened to expose the heart, and the ventricles were perfused with 4% paraformaldehyde solution at 4°C. Successful perfusion was indicated by stiffness of the limbs and tail. The animals were immediately decapitated and the brain was removed. The extracted brain tissue specimen was then immersed in 4% paraformaldehyde solution and fixed at 4°C for 12 hours. Dehydration was performed using a gradient of 20% and 30% sucrose solutions. Coronal sections were cut to 25µm using a cryostat, and brain slices were stored in in situ hybridization protection solution at -20°C for later use.

[0042] 1.4 Immunofluorescence staining:

[0043] (1) Rinse the cut brain tissue slices in a solution of 0.01M PBS for 5 min × 3 times;

[0044] (2) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0045] (3) 1% 0.01M PBST was transfused into the membrane at room temperature for 15 min;

[0046] (4) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0047] (5) Block with 10% serum at room temperature for 1 hour;

[0048] (6) Prepare the GFAP primary antibody mixed working solution according to the ratio and incubate overnight at 4 degrees Celsius;

[0049] (7) Rinse with 0.3% 0.01M PBST for 5 min × 3 times.

[0050] (8) Transfer the cleaned specimen into the prepared secondary antibody working solution and incubate at room temperature for 1 hour;

[0051] (9) Rinse with 0.3% 0.01M PBST for 10 min × 3 times;

[0052] (10) Lay the brain slices flat on a clean glass slide and pat them dry with absorbent paper. Then add DAPI-containing mounting medium and cover with a coverslip.

[0053] (11) Select a suitable field of view for observation under a laser confocal microscope and acquire images.

[0054] (12) The acquired images were analyzed using Image J image analysis software. After setting the fluorescence threshold, the co-labeling status of two fluorescence types in the periventricular white matter was determined, and the data were analyzed.

[0055] 1.5 Staggered Step Experiment:

[0056] Before modeling, mice were trained for 3 days, 5 minutes each day, to acclimatize them to walking on a wire mesh. On the day of modeling, and on days 7, 14, and 28 after modeling, 3-minute videos of the mice walking on the wire mesh were recorded. The total number of steps and the number of missteps in the last minute were counted. Misstep rate = (Number of missteps / Total steps) x 100%.

[0057] The results are as follows Figure 1 As shown, compared with the model group, only the 0.5 g / kg ManNAc treatment group significantly reduced both the misstep rate and the area of ​​GFAP-positive regions. Therefore, 0.5 g / kg was used as the dosage concentration of ManNAc.

[0058] 2. Effects of ManNAc on astrocyte proliferation and polarization in a mouse model of hydrocephalus:

[0059] Seven days after modeling hydrocephalus, mice were subjected to immunofluorescence staining for GFAP / C3d and GFAP / S100A10. GFAP is a molecular marker of astrocyte proliferation, while C3d and S100A10 are molecular markers of astrocyte polarization. Results are as follows: Figure 2Image A, Image CE, Area of ​​GFAP-positive region in periventricular white matter of mice in ManNAc treatment group, GFAP + C3d + The proportion of cells was significantly lower than that in the model group, and GFAP + S100A10 + The proportion of cells was significantly higher than that in the model group. Simultaneously, the periventricular white matter region of the brain tissue was collected from the hydrocephalus model mice 7 days after modeling, and the expression levels of GFAP, C3d, and S100A10 proteins were detected by Western blotting. The results are as follows: Figure 2 Figure B and Figure FH show that the expression levels of GFAP and C3d proteins in the ManNAc treatment group were significantly lower than those in the model group, while the expression level of S100A10 protein was significantly higher. This indicates that ManNAc administration can promote the proliferation of astrocytes in mice with hydrocephalus and promote the conversion of astrocytes from the characteristic A1 type (neurotoxic type) expressing C3d to the characteristic A2 type (neuroprotective type) expressing S100A10.

[0060] Detailed experimental procedure:

[0061] 2.1 Frozen Sections of Brain Tissue: After drug administration on day 7, experimental animals were perfused and fixed. Anesthetized with pentobarbital via intraperitoneal injection, the thoracic cavity was opened to expose the heart, and the ventricles were perfused with 4% paraformaldehyde solution at 4°C. Successful perfusion was indicated by stiffness of the limbs and tail. The animals were immediately decapitated and the brain was removed. The extracted brain tissue specimen was then immersed in 4% paraformaldehyde solution and fixed at 4°C for 12 hours. Dehydration was performed using a gradient of 20% and 30% sucrose solutions. Coronal sections were cut to 25µm using a cryostat, and brain slices were stored in in situ hybridization protection solution at -20°C for later use.

[0062] 2.2 Immunofluorescence staining:

[0063] (1) Rinse the cut brain tissue slices in a solution of 0.01M PBS for 5 min × 3 times;

[0064] (2) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0065] (3) 1% 0.01M PBST was transfused into the membrane at room temperature for 15 min;

[0066] (4) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0067] (5) Block with 10% serum at room temperature for 1 hour;

[0068] (6) Prepare a mixed working solution of GFAP / C3d or GFAP / S100A10 primary antibody according to the ratio and incubate overnight at 4 degrees Celsius.

[0069] (7) Rinse with 0.3% 0.01M PBST for 5 min × 3 times.

[0070] (8) Transfer the cleaned specimen into the prepared secondary antibody working solution and incubate at room temperature for 1 hour;

[0071] (9) Rinse with 0.3% 0.01M PBST for 10 min × 3 times.

[0072] (10) Lay the brain slices flat on a clean glass slide and dab them dry with absorbent paper. Then add a sealing agent and cover with a glass slide.

[0073] (11) Select a suitable field of view for observation under a laser confocal microscope and acquire images.

[0074] (12) The acquired images were analyzed using Image J image analysis software. After setting the fluorescence threshold, the co-labeling status of two fluorescence types in the periventricular white matter was determined, and the data were analyzed.

[0075] The results are as follows Figure 2 As shown in Figure A and Figure CE, the area of ​​the GFAP-positive region in the periventricular white matter of mice in the ManNAc treatment group, and the GFAP... + C3d + The proportion of cells was significantly lower than that in the model group, and GFAP + S100A10 + The proportion of cells was significantly higher than that in the model group.

[0076] 2.3 Brain tissue protein extraction:

[0077] (1) After administration on day 7, the experimental animals were perfused and fixed. The animals were anesthetized by intraperitoneal injection of pentobarbital, and the thoracic cavity was opened to expose the heart. The ventricles were perfused with 4°C physiological saline. The animals were immediately decapitated and the brain was removed. The periventricular tissue was then separated from the removed brain tissue specimen and stored at -80°C for later use.

[0078] (2) Place the excised brain tissue into the prepared protein lysis extraction solution (add protease inhibitor and phosphorylase inhibitor to the protein lysis solution in advance), and homogenize and dissolve the brain tissue thoroughly on a homogenizer.

[0079] (3) After thorough homogenization of the brain tissue, it was placed in an ice box for further lysis for 15 minutes. After lysis, it was centrifuged at 4°C.

[0080] Centrifuge for 15 minutes at a speed of 12,000 rpm;

[0081] (4) After centrifugation, use a pipette to remove the supernatant from the centrifuge tube;

[0082] (5) The protein content of the sample was then detected using a BCA kit to determine the protein concentration.

[0083] (6) Prepare SDS-PAGE gels of a certain concentration according to the instructions based on the molecular weight of the proteins to be tested, and aspirate the gels containing...

[0084] Add bromophenol blue loading buffer to a 30ug protein sample, incubate in a 95°C metal bath for 5 minutes, and then perform SDS-PAGE gel electrophoresis.

[0085] (7) After electrophoresis, the proteins on the gel were transferred to a cellulose nitrate membrane using a wet transfer method under constant voltage of 70V for 110 minutes.

[0086] (9) Then immerse the nitrocellulose membrane in 5% skim milk powder solution and seal for 1 hour, then shake slowly on a shaker at room temperature;

[0087] (10) Wash the nitrocellulose membrane with PBST buffer (0.01M) containing 0.1% Tween-20 for 10 min x 3 times;

[0088] (11) Dilute GFAP, C3d, S100A10 and GAPDH antibodies with primary antibody dilution solution according to the ratio, then immerse the nitrocellulose membrane in the antibody dilution solution respectively, and place the antibody incubation box on a shaker at 4 degrees and shake slowly overnight;

[0089] (12) The nitrocellulose membrane was washed with PBST buffer (0.01M) containing 0.1% Tween-20 for 10 minutes × 3 times;

[0090] (13) Dilute the corresponding secondary antibody with diluent, immerse the nitrocellulose membrane in the secondary antibody solution and incubate for 1 hour, then shake slowly on a shaker at room temperature;

[0091] (14) The nitrocellulose membrane was washed with PBST buffer (0.01M) containing 0.1% Tween-20 for 10 minutes × 3 times;

[0092] (15) Prepare the ECL color development solution (A solution: B solution = 1:1), put the nitrocellulose membrane into the chemiluminescent gel imaging instrument, and use the ECL color development solution to perform color development, exposure and image acquisition;

[0093] (16) The relative density of the collected images was determined using Image Lab image analysis software, and the data was analyzed.

[0094] The results are as follows Figure 2As shown in Figures B and FH, the expression levels of GFAP and C3d proteins in the ManNAc treatment group were significantly lower than those in the model group, while the expression level of S100A10 protein was significantly higher. This indicates that ManNAc administration can promote the proliferation of astrocytes in mice with hydrocephalus and promote the conversion of astrocytes from the characteristic A1 type (neurotoxic type) expressing C3d to the characteristic A2 type (neuroprotective type) expressing S100A10.

[0095] 3. Protection of periventricular white matter demyelination injury in a mouse model of hydrocephalus:

[0096] After the initial drug administration, the experimental animals were kept in the hospital for 35 days. Mice were then sacrificed, and brain sections were obtained via perfusion for MBP immunofluorescence staining. MBP is an important molecule constituting the myelin sheath of brain white matter. Results are as follows... Figure 3 As shown, the MBP staining fluorescence intensity of the cingulum bundle (CG) and external capsule (EC) in the periventricular white matter region of the ManNAc treatment group was significantly higher than that of the model group, indicating that ManNAc administration has a protective effect against periventricular white matter demyelination injury caused by hydrocephalus.

[0097] Detailed experimental procedure:

[0098] 3.1 Frozen Sections of Brain Tissue: After drug administration on day 14, animals were kept for 35 days post-modeling. The animals were then subjected to perfusion fixation. Anesthesia was administered via intraperitoneal injection of pentobarbital. The thoracic cavity was opened to expose the heart. Ventricular perfusion was performed using 4% paraformaldehyde solution at 4°C. Successful perfusion was indicated by stiffness of the limbs and tail. The animals were immediately decapitated and the brain was removed. The extracted brain tissue specimen was then immersed in 4% paraformaldehyde solution and fixed at 4°C for 12 hours. Dehydration was performed using a gradient of 20% and 30% sucrose solutions. Coronal sections (25 μm) were prepared using a cryostat. Brain slices were placed in in situ hybridization protection solution and stored at -20°C for later use.

[0099] 3.2 Immunofluorescence staining:

[0100] (1) Rinse the cut brain tissue slices in a solution of 0.01M PBS for 5 min × 3 times;

[0101] (2) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0102] (3) 1% 0.01M PBST was transfused into the membrane at room temperature for 15 min;

[0103] (4) Rinse with 0.3% 0.01M PBST for 5 min × 3 times;

[0104] (5) Block with 10% serum at room temperature for 1 hour;

[0105] (6) Prepare the MBP primary antibody mixed working solution according to the ratio and incubate overnight at 4 degrees Celsius;

[0106] (7) Rinse with 0.3% 0.01M PBST for 5 min × 3 times.

[0107] (8) Transfer the cleaned specimen into the prepared secondary antibody working solution and incubate at room temperature for 1 hour;

[0108] (9) Rinse with 0.3% 0.01M PBST for 10 min × 3 times.

[0109] (10) Lay the brain slices flat on a clean glass slide and dab them dry with absorbent paper. Then add a sealing agent and cover with a glass slide.

[0110] (11) Select a suitable field of view for observation under a laser confocal microscope and acquire images.

[0111] (12) The fluorescence intensity of the periventricular white matter was measured after setting the fluorescence threshold using the image analysis software ImageJ, and the data were analyzed.

[0112] The results are as follows Figure 3 As shown, the MBP staining fluorescence intensity of the cingulum bundle (CG) and external capsule (EC) in the periventricular white matter region of the ManNAc treatment group was significantly higher than that of the model group, indicating that ManNAc administration has a protective effect against periventricular white matter demyelination injury caused by hydrocephalus.

[0113] 4. The effect of ManNAc on reducing periventricular hyperintensity on magnetic resonance imaging in a mouse model of hydrocephalus:

[0114] Twenty-eight days after modeling, the experimental animals underwent cranial scanning using 11.7T high-field small animal MRI. After induction anesthesia with 1%-2% isoflurane, the animals were fixed in the incisor groove of the coil and continuously anesthetized with 1.5% isoflurane. Whole-brain coronal MRI T2-weighted sequences were performed at a slice thickness of 500 μm. The Evans ratio and the area of ​​periventricular high signal intensity at corresponding levels were calculated using the acquired T2-series images. The Evans ratio is calculated as the ratio of the width of the widest level of the bilateral lateral ventricles to the width of the widest level of the entire brain, and it can be used to measure ventricular size. The area of ​​periventricular high signal intensity was measured using ImageJ software.

[0115] The results are as follows Figure 4As shown, although the ventricular size of mice in the ManNAc treatment group was similar to that in the model group, the periventricular high signal intensity was significantly reduced. This indicates that while ManNAc administration cannot reverse ventricular enlargement caused by hydrocephalus, it can alleviate the white matter high signal intensity secondary to ventricular enlargement.

[0116] 5. Protection of motor dysfunction in a mouse model of hydrocephalus using ManNAc:

[0117] Motor function of the hydrocephalus model mice was assessed continuously on days 7, 14, and 28 after modeling. The staggered gait test was used to assess motor coordination, the robin test to assess balance, and Catwalk gait analysis to assess gait abnormalities. Figure 5 As shown in Figure A, the time mice in the ManNAc treatment group spent on the rotarod was significantly prolonged, with statistically significant differences at 14 and 28 days after modeling. Figure 5 As shown in Figure B, compared with the model group, the misstep rate of mice in the ManNAc treatment group was reduced, and there were significant statistical differences at 7, 14 and 28 days after modeling. Figure 5 As shown in Figure C1, the severity of gait abnormalities such as shortened stride length, bradygait, and balance disorder in the hydrocephalus model mice significantly decreased after ManNAc administration. Compared to the model group, the ManNAc treatment group showed increased stride length (Figure D), increased speed (Figure E), shortened step duration (Figure F), reduced standing time (Figure G), increased steps per minute (Figure H), and improved diagonal support ratio (Figure I). ManNAc significantly improved the movement speed of the mice as early as day 7 after modeling, while improvements in other indicators appeared after day 14. This indicates that ManNAc administration can significantly improve motor disorders in the hydrocephalus model mice, including motor coordination, balance impairment, and gait abnormalities.

[0118] Detailed experimental procedure:

[0119] 5.1 Rotary Test: Three days of pre-modeling training were conducted. Mice were placed on a rotarod fatigue tester. On the first day, the rotarod was rotated at 5 rpm for 300 seconds. On the second and third days, the rotation speed was constantly increased from 5 rpm to 40 rpm for a total of 300 seconds, allowing the mice to adapt to the rotarod. On the day of modeling, and on days 7, 14, and 28 after modeling, the rotation speed was constantly increased from 5 rpm to 40 rpm for a total of 300 seconds, and the time it took for the mice to fall was recorded. This was performed three times a day, with a 10-minute interval between each test.

[0120] like Figure 5 As shown in Figure A, the time mice in the ManNAc treatment group spent on the rotarod was significantly prolonged, with statistically significant differences at 14 and 28 days after modeling.

[0121] 5.2 Misstepping Experiment: Mice were trained for 3 days prior to modeling, with 5 minutes of walking time each day, to allow them to adapt to walking on a wire mesh. On the day of modeling, and on days 7, 14, and 28 after modeling, 3-minute videos of mice walking on the wire mesh were recorded. The total number of steps and the number of missed steps in the last minute were counted. Misstepping rate = (Number of missteps / Total steps) x 100%.

[0122] like Figure 5 As shown in Figure B, compared with the model group, the misstep rate of mice in the ManNAc treatment group was reduced, and there were significant statistical differences at 7, 14 and 28 days after modeling.

[0123] 5.3 Catwalk Gait Analysis: Three days before modeling, mice were placed on the Catwalk gait analysis system track to allow them to acclimatize. A mouse was considered successfully trained if it could traverse the track three times without stopping in the same direction. On days 7, 14, and 28 after modeling, mice were placed on one side of the track and allowed to traverse freely in one direction without interference. Green footprints appeared on the track during the mouse's running, simultaneously captured and identified by a camera. For each mouse, at least three records showed a maximum speed variability of less than 30%. Using CatWalk XT 10.6 software, gait parameters such as stride length, speed, step cycle, duty cycle, steps per minute (cadence), and support diagonal were analyzed and exported.

[0124] like Figure 5 As shown in Figure C1, the severity of gait abnormalities, including shortened stride length, bradygait, and balance disorder, in the hydrocephalus model mice significantly decreased after ManNAc administration. Compared to the model group, the ManNAc treatment group showed increased stride length (Figure D), increased speed (Figure E), shortened step duration (Figure F), reduced standing time (Figure G), increased steps per minute (Figure H), and improved diagonal support ratio (Figure I). ManNAc significantly improved the movement speed of the mice as early as day 7 after modeling, while improvements in other indicators appeared after day 14. This indicates that ManNAc administration can significantly improve motor disorders in the hydrocephalus model mice, including motor coordination, balance impairment, and gait abnormalities.

[0125] 6. Protective effect of ManNAc on long-term cognitive impairment in a mouse model of hydrocephalus:

[0126] After the initial drug administration, the experimental animals were kept in captivity for 28 days. Following a 4-day acclimatization period starting on day 28, training for object recognition was conducted on day 5, and testing on day 6. The exploration time for each object was recorded on both days. Based on the recorded exploration time, the differential index (DI) was calculated for both the training and testing phases. DI = (Time spent exploring the new object – Time spent exploring the familiar object) / (Time spent exploring the new object # Time spent exploring the familiar object) × 100%. The difference between the DI in the testing phase and the DI in the training phase was used as the preference index (PI), which measures the animal's spatial memory ability. Results are as follows: Figure 6 As shown, the PI of the ManNAc treatment group was larger than that of the model group, indicating that ManNAc administration can significantly improve the spatial memory ability of hydrocephalus model mice, thereby reducing the cognitive impairment caused by long-term hydrocephalus.

[0127] Detailed experimental procedure:

[0128] After the experimental animals finished receiving the drug, they were kept in the environment for 28 days. For the next three days, they were placed in a circular black barrel (26cm in diameter, 38cm high) for 30 minutes each day, and on the fourth day, for 10 minutes, to allow them to acclimatize. Day 5 was the training phase, where the animals were trained to explore and remember two objects 20cm apart within 6 minutes. Day 6 was the testing phase, where one of the objects was replaced with a novel object of a different shape and color, and the animals were tested on their exploration within 6 minutes. The exploration time for the two objects was recorded on both days 5 and 6. Based on the recorded exploration time, the differential index (DI) was calculated for both the training and testing phases. DI = (Time spent exploring the new object – Time spent exploring the familiar object) / (Time spent exploring the new object # Time spent exploring the familiar object) × 100%. The difference between the DI in the testing phase and the DI in the training phase is the preference index (PI), used to measure the animal's spatial memory ability.

[0129] The results are as follows Figure 6 As shown, the PI of the ManNAc treatment group was larger than that of the model group, indicating that ManNAc administration can significantly improve the spatial memory ability of hydrocephalus model mice, thereby reducing the cognitive impairment caused by long-term hydrocephalus.

[0130] The above results indicate that in a mouse model of hydrocephalus, ManNAc can inhibit astrocyte proliferation and neurotoxic polarization during the acute phase; and alleviate white matter myelin damage in the chronic phase, thereby reducing periventricular hyperintensity on magnetic resonance imaging, ultimately improving motor dysfunction and long-term cognitive impairment caused by hydrocephalus. Therefore, ManNAc has been shown to be a potential drug for treating neurological dysfunction caused by hydrocephalus.

[0131] Applications of this invention:

[0132] When preparing ManNAc into a drug, an effective amount of ManNAc can be formulated together with at least one pharmaceutically acceptable carrier, diluent, or excipient. In preparing these compositions, the active ingredient is typically mixed with, diluted with, or encapsulated in a carrier that may be in capsule or sac form. When the excipient acts as a diluent, it can be a solid, semi-solid, or liquid material serving as the medium for the excipient, carrier, or active ingredient. Therefore, the dosage form can be a liquid, solid, or semi-solid dosage form. Liquid dosage forms can include solutions (including true solutions and colloidal solutions), emulsions (including O / W, W / O, and double emulsions), suspensions, injections (including aqueous injections, powder injections, and infusions), eye drops, nasal drops, lotions, and liniments, etc.; solid dosage forms can include tablets (including regular tablets, enteric-coated tablets, lozenges, dispersible tablets, chewable tablets, effervescent tablets, and orally disintegrating tablets), capsules (including hard capsules, soft capsules, and enteric-coated capsules), granules, powders, microcapsules, pellets, suppositories, films, patches, aerosols, and sprays, etc.; semi-solid dosage forms can include ointments, gels, and pastes, etc. Suitable excipients include: lactose, glucose, sucrose, sorbitol, mannitol, starch, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, etc. Preparations may also include: humectants, emulsifiers, preservatives (such as methylparaben and propylparaben), sweeteners, etc.

[0133] like Figure 1 The figure shown is a graph illustrating the results of a concentration gradient experiment of ManNAc in a mouse model of hydrocephalus.

[0134] Figure A shows the GFAP / DAPI immunofluorescence double staining pattern, with a scale bar of 20 μm. Figure B shows the baseline on the day of modeling and the results of the staggered step test on day 7 after modeling. Figure C shows the statistical results of the percentage of GFAP+ positive areas around the ventricles.

[0135] In the figure, Ctr+Veh represents the control group; HCP+Veh represents the model group; 0.25 represents the 0.25 g / kg ManNAc treatment group; 0.5 represents the 0.5 g / kg ManNAc treatment group; 1 represents the 1 g / kg ManNAc treatment group; *, P<0.05; **, P<0.01; ***, P<0.001.

[0136] like Figure 2 The figure shown is a graph illustrating the effects of ManNAc on astrocyte proliferation and polarization in a mouse model of hydrocephalus.

[0137] Figure A shows the immunofluorescence double staining images of GFAP / C3d and GFAP / S100A10, with a scale bar of 20 μm. Figure B shows the Western blot results of GFAP, C3d, and S100A10 protein expression in the periventricular white matter. Figures C, D, and E show the results of GFAP in the periventricular white matter. + Percentage of positive areas, GFAP + C3d + Cells and GFAP + S100A10 + Cells account for GFAP + Cell proportion statistics. Figures F, G, and H show the relative expression levels of GFAP, C3d, and S100A10 proteins P compared to GAPDH in the periventricular region of the brain.

[0138] In the figure, Ctr+Veh is the control group; HCP+Veh is the model group; HCP+ManNAc is the ManNAc treatment group; *, P<0.05; **, P<0.01; ***, P<0.001.

[0139] like Figure 3 The figure shown is a diagram illustrating the effect of ManNAc on periventricular white matter demyelination damage in a hydrocephalus model mouse.

[0140] Figure A shows the immunofluorescence staining of the cingulum bundle (CG) and external capsule (EC) MBP in the periventricular white matter, with a scale bar of 40 μm. Figures B and C show the statistical graphs of the fluorescence intensity of the cingulum bundle (CG) and external capsule (EC) MBP in the periventricular white matter.

[0141] In the figure, Ctr+Veh is the control group; HCP+Veh is the model group; HCP+ManNAc is the ManNAc treatment group; *, P<0.05; **, P<0.01; ***, P<0.001.

[0142] like Figure 4 The image shows the effect of ManNAc on periventricular high signal on MRI in a mouse model of hydrocephalus.

[0143] Image A is a T2-weighted magnetic resonance imaging (MRI) coronal scan of the head; Image B is a statistical chart of Evan's index; Image C is a statistical chart of the area of ​​high signal intensity in the periventricular region.

[0144] In the figure, Ctr+Veh represents the control group; HCP+Veh represents the model group; and HCP+ManNAc represents the ManNAc treatment group; ***, P<0.001; ns, not significant;

[0145] like Figure 5 The figure shown is a graph illustrating the effect of ManNAc on motor dysfunction in a hydrocephalus model mouse.

[0146] Figure A shows the dwell time in the rotarod experiment on the day of modeling and at 7, 14, and 28 days after modeling; Figure B shows the misstep rate in the misstep experiment on the day of modeling and at 7, 14, and 28 days after modeling; Figure C shows representative gait footprints of the two groups of mice recorded by the Catwalk gait analysis system on day 28 after modeling. Figures D-I show various gait parameters of the two groups of mice analyzed by the Catwalk gait analysis system, including stride length (Figure D), speed (Figure E), stride duration (Figure F), the proportion of standing time in each stride (Figure G), strides per minute (Figure H), and diagonal support ratio (Figure I).

[0147] In the figure, Ctr+Veh represents the control group; HCP+Veh represents the model group; and HCP+ManNAc represents the ManNAc treatment group. *, P<0.05; **, P<0.01; ***, P<0.001; ###, P<0.001

[0148] like Figure 6 The figure shown is a graph illustrating the effect of ManNAc on long-term cognitive impairment in a hydrocephalus model mouse.

[0149] Figure A shows the representative movement trajectories of the two groups of mice obtained during the testing phase of the new object recognition experiment; Figure B shows the difference in preference index (PI) between the testing and training phases of the new object recognition experiment.

[0150] In the figure, Ctr+Veh is the control group; HCP+Veh is the model group; HCP+ManNAc is the ManNAc treatment group; *, P<0.05.

[0151] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. The use of N-acetylmnosamine in the preparation of drugs for the treatment or prevention of neurological dysfunction caused by hydrocephalus.

2. The application according to claim 1, characterized in that, The dosage forms of the drug include tablets, powders, granules, capsules, oral liquids, injections, or sustained-release formulations.

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