CN116407546A - Application of N-acetylmannosamine in preparation of medicines - Google Patents
Application of N-acetylmannosamine in preparation of medicines Download PDFInfo
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- CN116407546A CN116407546A CN202310369145.8A CN202310369145A CN116407546A CN 116407546 A CN116407546 A CN 116407546A CN 202310369145 A CN202310369145 A CN 202310369145A CN 116407546 A CN116407546 A CN 116407546A
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- hydrocephalus
- mannac
- acetylmannosamine
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- medicament
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Abstract
The invention relates to an application of N-acetylmannosamine in preparation of medicines, and belongs to the technical field of biological medicines. The invention provides an application of N-acetylmannosamine in preparing a medicament for treating or preventing hydrocephalus-induced nerve dysfunction; the application in preparing the medicine for treating or preventing dyskinesia caused by hydrocephalus and neurodegenerative diseases; use in the manufacture of a medicament for improving cognitive dysfunction; application in preparing medicines for inhibiting astrocyte proliferation and promoting astrocyte transformation; and the application in preparing medicines for relieving the white matter demyelinating injury of brain tissues. By using the ManNAc provided by the invention, a new medicine is provided for preventing and treating the neurological dysfunction caused by hydrocephalus, and a new treatment direction is provided for patients.
Description
Technical Field
The invention relates to an application of N-acetylmannosamine in preparation of medicines, and belongs to the technical field of biological medicines.
Background
Hydrocephalus refers to an expansion of the ventricle caused by an obstruction in the process of cerebrospinal fluid production or circulatory absorption. Hydrocephalus can be classified into primary and secondary types according to the etiology. Primary hydrocephalus is due to congenital or genetic abnormalities in cerebrospinal fluid circulation or absorption, such as congenital ventricular stenosis, post-meningitis syndrome, and the like. Secondary hydrocephalus is a disorder of cerebrospinal fluid circulation or absorption caused by other diseases or trauma, such as brain tumor, cerebral hemorrhage, brain trauma, etc. In addition, neurodegenerative diseases such as Alzheimer's disease and the like, parkinson's disease, amyotrophic lateral sclerosis and the like can also cause atrophy of cerebral cortex, which in turn causes ventricular dilatation.
Hydrocephalus, including expansion of the ventricle secondary to neurodegenerative diseases, can cause collateral damage to the ventricle, abnormal proliferation and polarization of glial cells, leading to a range of neurological disorders including dyskinesia, cognitive disorders, vision disorders, and the like. Among them, dyskinesias are particularly important in the academia. Dyskinesias such as hypomyosis, impaired motor balance and coordination, involuntary movement of muscles and the like seriously affect the daily life of a patient, and a series of risks endangering the life of the patient such as falling can be generated.
Hydrocephalus has become a serious public health problem in recent years, and has taken an increasingly important role in the human disease spectrum. It is counted that idiopathic normal craniocerebral pressure hydrocephalus (a type of hydrocephalus common to the elderly) accounts for 0.5% -1.5% of the population over 60 years of age in our country. While patients of various other etiologies are about 2-3 times that of idiopathic normal craniocerebral pressure hydrocephalus.
However, the treatment of hydrocephalus is currently facing a great challenge, and there is a lack of therapeutic approaches that can effectively delay the progression of the disease. Except for removing the primary cause, the cerebrospinal fluid shunt operation is a first-line treatment means of hydrocephalus, and concretely comprises ventricular and peritoneal drainage, ventricular and atrial drainage, lumbar and greater pool peritoneal drainage and the like. However, there is still some controversy over current surgical treatments for hydrocephalus. First, the long-term curative effect of the operation is doubtful, and long-term follow-up researches show that the curative effect of the patient can be gradually reduced along with time due to occlusion of the drainage catheter, and about 30-50% of patients need to be operated again. Secondly, the operation is invasive, the occurrence rate of complications such as subdural hematoma, infection and the like is high, and the elderly patients with serious basic diseases are difficult to bear. Therefore, exploration of drug intervention strategies for hydrocephalus is imperative.
Neu5Ac is a natural saccharide compound with nine carbon sugars as a basic skeleton, and is one of the most common sialic acids in humans. The Neu5Ac has few free forms and plays an important role in mediating cell-to-cell recognition, adhesion, migration and the like by being involved in forming cell glycoprotein and glycolipid terminal sugar chains in vivo. In the nervous system, neu5Ac is present in relatively high levels, which is statistically about 20 times the average levels of other system tissues. Neu5Ac of brain tissue is mainly involved in constituting gangliosides and glycoproteins, and plays very unique and key roles in the aspects of synapse formation, nerve impulse transmission, maintenance of neuronal and myelin function homeostasis, and the like. Studies have shown that the lack of Neu5Ac and its complexes can lead to a series of clinical symptoms similar to hydrocephalus, such as reduced motor function, cognitive dysfunction, etc. While exogenous supplementation of N-acetylmannosamine (ManNAc), a precursor to the Neu5Ac synthesis pathway, partially reverses clinical symptoms. Therefore, manNAc is expected to play an important role in the prevention or treatment of hydrocephalus dyskinesia. As a precursor for the synthesis of Neu5Ac molecules, manNAc is an uncharged monosaccharide with a molecular weight of 221 daltons. ManNAc has been shown to have potential effects in the treatment of dyskinesias caused by some neuromuscular diseases. For example, GNE myopathy is an autosomal recessive genetic disease caused by mutation of the GNE gene, which encodes a rate-limiting enzyme important in Neu5Ac synthesis. Due to the defect of the GNE enzyme, the Neu5Ac content in muscle tissues of patients is obviously reduced, so that sugar chains are insufficient in sialylation, and symptoms such as muscle weakness and the like appear. Animal and clinical experiments show that the supplement of ManNAc can effectively improve the sialylation of the sugar chains of the muscle cells and improve dyskinesia. In addition, the ManNAc also has a certain protection effect on cognitive dysfunction, and can delay the cognitive dysfunction of middle-aged and old people by improving spatial memory, working memory and abnormal sleep rhythm. However, manNAc has not been effectively used in the current treatment of dyskinesias caused by hydrocephalus, including dyskinesias associated with neurodegenerative diseases.
Disclosure of Invention
In view of the lack of effective treatment means for the existing neurological dysfunction caused by hydrocephalus, especially dyskinesia, the invention aims to solve the technical problem of application of ManNAc in preparing medicaments for treating or preventing the neurological dysfunction caused by hydrocephalus.
In order to achieve the aim of the invention, the invention provides the application of N-acetylmannosamine in preparing a medicament for treating or preventing the neurological dysfunction caused by hydrocephalus.
The invention provides an application of N-acetylmannosamine in preparing a medicament for treating or preventing dyskinesia caused by hydrocephalus and neurodegenerative diseases.
The invention provides an application of N-acetylmannosamine in preparing a medicament for improving cognitive dysfunction.
The invention provides an application of N-acetylmannosamine in preparing a medicament for inhibiting astrocyte proliferation and promoting astrocyte transformation.
The invention provides an application of N-acetylmannosamine in preparing a medicine for relieving white matter demyelinating injury of brain tissue.
Preferably, the dosage forms of the medicament in the application comprise tablets, powder, granules, capsules, oral liquid, injection or sustained release agents and the like.
Compared with the prior art, the invention has the following beneficial effects:
ManNAc is an important precursor in the endogenous Neu5Ac biosynthesis pathway, and can efficiently synthesize Neu5Ac in cells. The molecule is neutral and is easy to be absorbed by cells. In addition, compared with the direct supplement of Neu5Ac, the supplement of ManNAc has long effective blood concentration maintenance time in animal experiments, and can effectively improve the concentration of Neu5Ac in the brain, which is helpful for the application of the medicine as a medicine for treating the neurological dysfunction caused by hydrocephalus.
From a safety point of view, a number of clinical trials concerning ManNAc treatment of neuromuscular diseases and glomerulonephritis have shown that they have no serious adverse effects at the oral administration dose, only a part of subjects report mild adverse effects of the gastrointestinal tract. The safety of the medicine is good.
From the viewpoint of effectiveness, the invention constructs an hydrocephalus animal model, and after ManNAc is used for the animal model, abnormal activation and polarization of astrocytes on pathological brain tissue of the model and white matter demyelination injury are inhibited; ventricular side elevation signal alleviation on magnetic resonance; the movement disorder and long-term cognitive disorder of the model are obviously improved. Therefore, the ManNAc has the medicinal effect of treating the nerve dysfunction caused by hydrocephalus.
The invention provides the application of ManNAc in preparing medicaments for treating or preventing the neurological dysfunction caused by hydrocephalus. The medicine has small molecular weight, can permeate the blood brain barrier, is easy to be absorbed by cells, has long effective blood concentration maintenance time, has small side effect on human bodies and has considerable clinical application prospect. The invention can be applied to clinic, and can be used as a novel medicament for preventing and treating the nerve dysfunction caused by hydrocephalus, thereby providing a novel treatment direction for patients.
Drawings
FIG. 1 is a graph showing the results of concentration gradient experiments of ManNAc administration in hydrocephalus model mice;
FIG. 2 is a graph showing the effect of ManNAc on proliferation and polarization of astrocytes in a hydrocephalus model mouse;
FIG. 3 is a graph showing the effect of ManNAc on periventricular white matter demyelination in mice with a hydrocephalus model;
FIG. 4 is a graph of the effect of ManNAc on the paracentricular elevation signal on the MRI of hydrocephalus model mice;
FIG. 5 is a graph of the effect of ManNAc on hydrocephalus model mouse dyskinesia;
FIG. 6 is a graph of the effect of ManNAc on long-term cognitive impairment in hydrocephalus model mice;
Detailed Description
In order to make the invention more comprehensible, preferred embodiments accompanied with the accompanying drawings are described in detail as follows:
the invention provides an application of N-acetylmannosamine in preparing a medicament for treating or preventing hydrocephalus-induced nerve dysfunction.
The invention provides an application of N-acetylmannosamine in preparing a medicament for treating or preventing dyskinesia caused by hydrocephalus and neurodegenerative diseases.
The invention provides an application of N-acetylmannosamine in preparing a medicament for improving cognitive dysfunction.
The invention provides an application of N-acetylmannosamine in preparing a medicament for inhibiting astrocyte proliferation and promoting astrocyte transformation.
The invention provides an application of N-acetylmannosamine in preparing a medicine for relieving white matter demyelinating injury of brain tissue.
The dosage forms of the medicine in the application comprise tablets, powder, granules, capsules, oral liquid, injection or sustained release agent and the like.
The molecular chemical formula of ManNAc is as follows:
the enlargement of the ventricles triggered by hydrocephalus and neurodegenerative diseases can lead to focal or extensive cerebral tissue blood hypoperfusion, which in turn leads to cellular ischemia and hypoxia, and reduced synthesis of endogenous Neu5Ac and its complexes, resulting in hyposialylation of various sugar chains on the tissue, leading to impairment of normal neurological functions, including motor functions. Therefore, supplementing Neu5Ac synthesis related substances, especially absorbable ManNAc, plays an important biological role in maintaining the steady state of a nervous system, relieving nerve dysfunction caused by hydrocephalus and the like.
Examples
1. Preparation and administration of animal models:
hydrocephalus model mice were constructed by a mouse occipital pool kaolin injection method (physiological saline was injected into the corresponding control group), and after molding, the model group was given subcutaneous injections of 0.3ml of ManNAc solution (the dose gradient of ManNAc was 0.25,0.5,1g/kg or 0.3ml physiological saline; the control group was given subcutaneous injections of 0.3ml physiological saline twice daily at 12 hours intervals for 14 consecutive days.
The specific experimental process comprises the following steps:
1.1 animal model preparation: after 27-30 g of adult male C57BL6 mice are selected and 1-2% isoflurane is used for inducing anesthesia, the mice are fixed on a stereotactic instrument, the angle of a mouth or an ear rod is adjusted, the head and the body form an angle of 120 degrees, and an anesthesia machine is started to continuously perform anesthesia by 1.5% isoflurane. Skin preparation of the occipital cell part, disinfection with iodophor, and peeling off subcutaneous soft tissue with sterile ophthalmic scissors to expose the occipital cell part. The hard membrane covering the occipital vat is wiped with an alcohol cotton swab. The syringe needle with the 30 gauge needle was then bent at 30-45 deg., and 10ul of 10% kaolin suspension (100 mg/ml in 0.9% saline) was withdrawn with the syringe. The needle was inserted into the occipital cell through the dura mater, the needle was slowly advanced 1.5-2.0mm, and 10ul of the kaolin suspension was slowly injected into the basal cell (about 1 ul/min). The needle was slowly withdrawn after leaving the needle for 5 minutes, and the surgical incision was sutured and sterilized after no reflux. The operation animals are placed in an electric incubator at 37 ℃ for resuscitation, and the operation animals are put back to a feeding platform for feeding after the experimental animals are revived. The experimental animals of the sham treatment group were injected with 10ul of sterile physiological saline in the same manner.
1.2 administration: immediately after the molding, the administration was performed in groups, and twice daily at 12-hour intervals. The control group and the model group were subcutaneously administered with 0.3ml of physiological saline/one at a time. The ManNAc treatment fraction was administered twice, each subcutaneously with a dose of 0.25,0.5,1 g/kg/dose of ManNAc in 0.3ml physiological saline. The three groups were each dosed for 14 days continuously.
1.3 frozen sections of brain tissue: after the end of the administration on day 7, the experimental animals were fixed by perfusion. Pentobarbital anesthesia was injected intraperitoneally, the chest of the experimental animal was opened to reveal the heart, ventricular infusion was performed with 4% paraformaldehyde solution at 4 ℃, and success of infusion was indicated when the experimental mouse had stiff limbs and tails. Immediately cutting the head to obtain brain, soaking the brain tissue sample in 4% paraformaldehyde solution, storing in a refrigerator at 4deg.C, and fixing for 12 hr. Sequentially changing 20% sucrose solution and 30% sucrose solution for gradient dehydration. The frozen microtome has a crown slice of 25um, and the brain slice is put into in situ hybridization protective solution to be preserved at-20 ℃ for standby.
1.4 immunofluorescent staining:
(1) Placing the cut brain tissue slice into a culture with 0.01M PBS, and rinsing for 5min×3 times;
(2) 0.3% of 0.01M PBST rinse 5min x3 times;
(3) 1% of 0.01M PBST is subjected to membrane penetration for 15min at room temperature;
(4) 0.3% of 0.01M PBST rinse 5min x3 times;
(5) Blocking for 1 hour at room temperature with 10% serum;
(6) Preparing GFAP primary antibody mixed working solution according to a proportion, and incubating overnight in a 4-DEG environment;
(7) 0.3% of 0.01M PBST rinse 5min x3 times.
(8) Transferring the cleaned specimen into a prepared corresponding secondary antibody working solution, and incubating for 1h at room temperature;
(9) 0.3% of 0.01M PBST rinse 10min x3 times;
(10) Spreading brain slice on clean glass slide, dipping with absorbent paper, dripping DAPI-containing sealing tablet, and covering with glass slide;
(11) And selecting a proper visual field for observation under a laser confocal microscope, and acquiring images.
(12) And (3) after the fluorescence threshold value is set by using the Image analysis software Image J, determining two fluorescence co-labeling conditions of white matter parts around the ventricle, and analyzing data.
1.5 misstep experiment:
training for 3 days before molding, 5min each day, and adapting the mice to walk on the wire mesh. On the day of molding, 7, 14, 28 days after molding, video of 3 minutes of mice walking on the wire netting is recorded, and the total walking steps and the stepping steps of the last minute are counted. Step rate = number of steps per total number of steps x100%.
The results are shown in figure 1, with only 0.5g/kg ManNAc treatment group, the step rate and GFAP positive area were significantly reduced compared to the model group. Thus, 0.5g/kg was used as the administration concentration of ManNAc.
Effect of mannac on proliferation and polarization of mouse astrocytes in a hydrocephalus model:
the hydrocephalus model mice 7 days after molding were stained for GFAP/C3d and GFAP/S100A10 immunofluorescence. GFAP is a molecular marker of astrocyte proliferation, while C3d and S100a10 are molecular markers of astrocyte polarization. The results are shown in FIG. 2, panel A, panel C-E, GFAP positive area and GFAP in the white matter region surrounding the ventricles of mice in the ManNAc treatment group + C3d + Cell proportion is obviously reduced compared with model group, GFAP + S100A10 + The proportion of cells was significantly increased compared to the model group. Meanwhile, a cerebral hydrops model mouse leaves a white matter area around a ventricle of brain tissue 7 days after modeling, and Western Blot detection is carried out on GFAP, C3d and S100A10 protein expression quantities. As shown in the B diagram and the F-H diagram of the figure 2, the expression level of GFAP and C3d proteins of mice in the ManNAc treatment group is obviously reduced compared with that of the model group, and the expression level of S100A10 protein is obviously increased compared with that of the model group. It was demonstrated that ManNAc administration promoted inhibition of astrocyte proliferation in mice from hydrocephalus model mice, and promoted conversion of astrocytes from type A1 (neurotoxic type) characterized by C3d expression to type A2 (neuroprotective type) characterized by S100A10 expression.
The specific experimental process comprises the following steps:
2.1 frozen sections of brain tissue: after the end of the administration on day 7, the experimental animals were fixed by perfusion. Pentobarbital anesthesia was injected intraperitoneally, the chest of the experimental animal was opened to reveal the heart, ventricular infusion was performed with 4% paraformaldehyde solution at 4 ℃, and success of infusion was indicated when the experimental mouse had stiff limbs and tails. Immediately cutting the head to obtain brain, soaking the brain tissue sample in 4% paraformaldehyde solution, storing in a refrigerator at 4deg.C, and fixing for 12 hr. Sequentially changing 20% sucrose solution and 30% sucrose solution for gradient dehydration. The frozen microtome has a crown slice of 25um, and the brain slice is put into in situ hybridization protective solution to be preserved at-20 ℃ for standby.
2.2 immunofluorescent staining:
(1) Placing the cut brain tissue slice into a culture with 0.01M PBS, and rinsing for 5min×3 times;
(2) 0.3% of 0.01M PBST rinse 5min x3 times;
(3) 1% of 0.01M PBST is subjected to membrane penetration for 15min at room temperature;
(4) 0.3% of 0.01M PBST rinse 5min x3 times;
(5) Blocking for 1 hour at room temperature with 10% serum;
(6) Preparing GFAP/C3d or GFAP/S100A10 primary antibody mixed working solution according to a proportion, and incubating overnight in a 4-DEG environment;
(7) 0.3% of 0.01M PBST rinse 5min x3 times.
(8) Transferring the cleaned specimen into a prepared corresponding secondary antibody working solution, and incubating for 1h at room temperature;
(9) 0.3% of 0.01M PBST rinse 10min x3 times;
(10) Spreading brain slice on clean glass slide, dipping with absorbent paper, dripping sealing tablet, and sealing with cover glass;
(11) And selecting a proper visual field for observation under a laser confocal microscope, and acquiring images.
(12) And (3) after the fluorescence threshold value is set by using the Image analysis software Image J, determining two fluorescence co-labeling conditions of white matter parts around the ventricle, and analyzing data.
The results are shown in FIG. 2, panel A, panel C-E, and the area of GFAP positive area in white matter region around the ventricles of mice in the ManNAc treatment group + C3d + Cell proportion is obviously reduced compared with model group, GFAP + S100A10 + The proportion of cells was significantly increased compared to the model group.
2.3 extraction of brain tissue proteins:
(1) After the end of the administration on day 7, the experimental animals were fixed by perfusion. Pentobarbital was intraperitoneally anesthetized, the chest of the experimental animal was opened to reveal the heart, and ventricular infusion was performed with physiological saline at 4 ℃. Immediately breaking the head, taking out the brain, separating the tissue around the ventricle from the taken out brain tissue specimen, and storing at-80 ℃ for standby.
(2) Placing the cut brain tissue into a protein lysis extracting solution prepared in advance (protease inhibitor and phosphorylase inhibitor are added into the protein lysis solution in advance), and fully homogenizing and dissolving the brain tissue on a homogenizer;
(3) After the brain tissue is sufficiently homogenized, the brain tissue is placed in an ice box for continuous lysis for 15min. After cleavage in a high-speed centrifuge at 4 DEG C
Centrifuging for 15min at 12000rpm;
(4) After centrifugation, absorbing supernatant of the centrifuge tube by using a pipettor;
(5) The protein content of the sample was then assayed for protein concentration using the BCA kit.
(6) According to the instruction, SDS-PAGE gel with a certain concentration is configured according to different molecular weights of proteins to be detected, and the gel is sucked to contain
Adding a sample to be detected of 30ug protein into a loading buffer solution containing bromophenol blue, carrying out metal bath at 95 ℃ for 5 minutes, and then carrying out SDS-PAGE gel electrophoresis;
(7) After electrophoresis, the proteins on the gel were transferred to a cellulose nitrate membrane using wet transfer at a constant pressure of 70V for 110 minutes.
(9) Then soaking the nitrocellulose membrane in 5% skimmed milk powder liquid for sealing for 1h, and slowly shaking on a shaking table at room temperature;
(10) The nitrocellulose membrane was washed 10minX3 times with PBST buffer (0.01M) containing 0.1% Tween-20;
(11) Diluting GFAP, C3d, S100A10 and GAPDH antibodies by using an anti-dilution solution in proportion, respectively soaking nitrocellulose membranes in the antibody dilution solution, and slowly shaking the antibody incubation box by a 4-DEG shaking table overnight;
(12) Nitrocellulose membranes were washed with PBST buffer (0.01M) containing 0.1% Tween-20 for 10min X3 times;
(13) Diluting the corresponding secondary antibody by using a diluent, soaking the nitrocellulose membrane in the secondary antibody liquid, incubating for 1h, and slowly shaking on a shaking table at room temperature;
(14) Nitrocellulose membranes were washed with PBST buffer (0.01M) containing 0.1% Tween-20 for 10min X3 times;
(15) Preparing ECL color developing solution (A solution: B solution=1:1), placing a nitrocellulose membrane into a chemiluminescent gel imaging instrument, and developing, exposing and collecting images by using the ECL color developing solution;
(16) The relative density of the collected pictures is measured by adopting Image Lab Image analysis software, and data are analyzed.
As shown in the B graph and the F-H graph of FIG. 2, the expression level of GFAP and C3d proteins of mice in the ManNAc treatment group is obviously reduced compared with that of the model group, and the expression level of S100A10 protein is obviously increased compared with that of the model group. It was demonstrated that ManNAc administration promoted inhibition of astrocyte proliferation in mice from hydrocephalus model mice, and promoted conversion of astrocytes from type A1 (neurotoxic type) characterized by C3d expression to type A2 (neuroprotective type) characterized by S100A10 expression.
Protection of periventricular white matter demyelinating lesions in mice with hydrocephalus model by mannac:
after the experimental animals were dosed, the animals were kept for 35 days, and the mice were sacrificed and perfused to obtain brain sections, and stained with MBP immunofluorescence. MBP is an important molecule constituting the myelin sheath of white matter. As shown in fig. 3, the fluorescence intensity of MBP staining of periventricular white matter area Cingulate (CG) and Exocyst (EC) of the ManNAc treatment group was significantly improved over that of the model group, demonstrating that ManNAc administration has a protective effect on periventricular white matter demyelination injury caused by hydrocephalus.
The specific experimental process comprises the following steps:
3.1 frozen sections of brain tissue: after the end of the administration on day 14, animals were kept until 35 days after molding, and experimental animals were fixed by perfusion. Pentobarbital anesthesia was injected intraperitoneally, the chest of the experimental animal was opened to reveal the heart, ventricular infusion was performed with 4% paraformaldehyde solution at 4 ℃, and success of infusion was indicated when the experimental mouse had stiff limbs and tails. Immediately cutting the head to obtain brain, soaking the brain tissue sample in 4% paraformaldehyde solution, storing in a refrigerator at 4deg.C, and fixing for 12 hr. Sequentially changing 20% sucrose solution and 30% sucrose solution for gradient dehydration. The frozen microtome has a crown slice of 25um, and the brain slice is put into in situ hybridization protective solution to be preserved at-20 ℃ for standby.
3.2 immunofluorescent staining:
(1) Placing the cut brain tissue slice into a culture with 0.01M PBS, and rinsing for 5min×3 times;
(2) 0.3% of 0.01M PBST rinse 5min x3 times;
(3) 1% of 0.01M PBST is subjected to membrane penetration for 15min at room temperature;
(4) 0.3% of 0.01M PBST rinse 5min x3 times;
(5) Blocking for 1 hour at room temperature with 10% serum;
(6) Preparing MBP primary antibody mixed working solution according to a proportion, and incubating overnight in a 4-DEG C environment;
(7) 0.3% of 0.01M PBST rinse 5min x3 times.
(8) Transferring the cleaned specimen into a prepared corresponding secondary antibody working solution, and incubating for 1h at room temperature;
(9) 0.3% of 0.01M PBST rinse 10min x3 times;
(10) Spreading brain slice on clean glass slide, dipping with absorbent paper, dripping sealing tablet, and sealing with cover glass;
(11) And selecting a proper visual field for observation under a laser confocal microscope, and acquiring images.
(12) And (3) after the fluorescence threshold value is set by using Image analysis software Image J, measuring the fluorescence intensity of white matter parts around the ventricle, and analyzing data.
As shown in fig. 3, the fluorescence intensity of MBP staining of periventricular white matter area Cingulate (CG) and Exocyst (EC) of the ManNAc treatment group was significantly improved over that of the model group, demonstrating that ManNAc administration has a protective effect on periventricular white matter demyelination injury caused by hydrocephalus.
Effect of mannac on reduction of ventricular side elevation signal on magnetic resonance in mice with hydrocephalus model:
experimental animals were scanned on the head of 11.7T high field small animals at magnetic resonance 28 days after molding. After induction of anesthesia with 1% -2% isoflurane, experimental animals were fixed on the coil door slot and anesthesia was continued with 1.5% isoflurane. The thickness of the scanning layer is 500um by scanning the whole brain coronal MRI T2 weighting sequence. And calculating Evans ratio and area of the corresponding level paraventricular high signal to the whole brain by using the acquired T2 sequence image. Evans ratio is calculated as the ratio of the width of the lateral ventricle at the widest level of the bilateral ventricle to the width of the widest level of the entire brain, which can be used to measure the ventricle size. The area of the whole brain occupied by the paraventricular high signal was measured using Image J software.
The results are shown in fig. 4, although the ventricle size of the ManNAc treated mice was similar to that of the model group, their paraventricular high signals were significantly reduced. This suggests that ManNAc administration does not reverse ventricular enlargement due to hydrocephalus, but reduces white matter high signals secondary to ventricular enlargement.
Protection of mannac against hydrocephalus model mouse dyskinesia:
the motor functions of hydrocephalus model mice were continuously assessed at days 7, 14, 28 post-molding. The step-by-step experiment is used for evaluating the movement coordination function of the mice, the rod rotating experiment is used for evaluating the balance capacity of the mice, and the Catwalk gait analysis is used for evaluating gait abnormality of the mice. As shown in figure 5, panel a, the residence time on the rotating bars was significantly prolonged for ManNAc treated mice, with significant statistical differences at 14, 28 days post-molding. As shown in panel B of fig. 5, the ManNAc treated mice had a reduced step rate compared to the model group, with significant statistical differences at 7, 14, and 28 days after molding. The extent of gait abnormalities such as shortened step size, retarded gait and imbalance in hydrocephalus model mice also decreased significantly after ManNAc administration, as shown in figures C-I of fig. 5. Compared to model groups, manNAc treated mice had longer step sizes (D panels), increased speeds (E panels), shorter cycles per step (F panels), reduced standing time scale (G panels), increased steps per minute (H panels), and increased diagonal support scale (I panels). On day 7 after molding, manNAc significantly increased the rate of movement of mice, while improvement in several other criteria occurred after day 14 of molding. This suggests that ManNAc administration may significantly improve dyskinesias including dyskinesias, impaired ability to balance, and gait abnormalities in mice with hydrocephalus models.
The specific experimental process comprises the following steps:
5.1 rotating rod experiment: training for 3 days before molding, placing the mice on a rotating rod fatigue instrument, rotating for 300s at a rotating speed of 5rpm in the first day, and enabling the rollers to be constantly accelerated from the rotating speed of 5rpm to 40rpm for 300s in the 2 nd and 3 rd days respectively, so that the mice adapt to rotating rods. On the day of molding, 7, 14, 28 days after molding, the rotational speed was constantly accelerated to 40rpm at 5rpm for 300 seconds in total, and the falling time of the mice was recorded. 3 times per day, each time interval of 10min.
As shown in figure 5, panel a, the residence time on the rotating bars was significantly prolonged for ManNAc treated mice, with significant statistical differences at 14, 28 days post-molding.
5.2 misstep experiment: training for 3 days before molding, 5min each day, and adapting the mice to walk on the wire mesh. On the day of molding, 7, 14, 28 days after molding, video of 3 minutes of mice walking on the wire netting is recorded, and the total walking steps and the stepping steps of the last minute are counted. Step rate = number of steps per total number of steps x100%.
As shown in panel B of fig. 5, the ManNAc treated mice had a reduced step rate compared to the model group, with significant statistical differences at 7, 14, and 28 days post-molding.
5.3Catwalk gait analysis: mice were placed on the racetrack of the Catwalk gait analysis system 3 days prior to molding to fit the environment. Mice passed the race track 3 times in the same direction without stopping can be regarded as being qualified for training. On days 7, 14, 28 after molding, mice were placed on one side of the racetrack and allowed to freely pass along the racetrack in one direction without interference. In the running process of the mice, green footprint marks appear on the runway and are captured and identified by the camera. Each mouse ensured a maximum speed variation of less than 30% for at least 3 recordings. Gait parameters such as Step length, speed, step period, standing time per Step (Duty cycle), step number per minute (Cadence), and diagonal Support ratio (Support diagonal) were derived using the CatWalk XT 10.6 software.
As shown in fig. 5, panels C-I, the extent of gait abnormalities such as shortening of the step, retardation of gait, and imbalance in hydrocephalus model mice also decreased significantly after ManNAc administration. Compared to model groups, manNAc treated mice had longer step sizes (D panels), increased speeds (E panels), shorter cycles per step (F panels), reduced standing time scale (G panels), increased steps per minute (H panels), and increased diagonal support scale (I panels). On day 7 after molding, manNAc significantly increased the rate of movement of mice, while improvement in several other criteria occurred after day 14 of molding. This suggests that ManNAc administration may significantly improve dyskinesias including dyskinesias, impaired ability to balance, and gait abnormalities in mice with hydrocephalus models.
Protection of ManNAc against long term cognitive impairment in hydrocephalus model mice:
the animals were kept for 28 days after the end of the administration. After 4 days of continuous adaptation from day 28, training of the new object recognition experiment was performed on day 5, testing of the new object recognition experiment was performed on day 6, and the exploration time of the object on days 5 and 6 was recorded, respectively. Based on the recorded exploration time, discrimination indices (differential index, DI) for the training and testing phases are calculated, respectively. Di= (time to search for new object side object-time to search for familiar object side object)/(time to search for new object side object # time to search for familiar object side object) ×100%. The difference between the test stage DI and the training stage DI is a Preference Index (PI) for measuring the spatial memory of the animal. The results are shown in fig. 6, which shows that ManNAc administration significantly improved spatial memory in hydrocephalus model mice compared to model groups, thereby reducing cognitive impairment resulting from long-term hydrocephalus.
The specific experimental process comprises the following steps:
the animals were kept on feeding for 28 days after the end of the administration, and the animals were placed in a round black tub with a diameter of 26cm and a height of 38cm for 30 minutes every day for the next 3 days, and placed in the black tub for 10 minutes on day 4, so as to be adapted to the environment. Day 5 is the training phase, where the training animal explores two objects with memory 20cm apart within 6 minutes. And the 6 th day is a testing stage, one of the novel objects with different shapes and colors is replaced, the exploration condition of the animal in 6 minutes is tested, and the exploration time of the 5 th day and the 6 th day for the two objects is recorded respectively. Based on the recorded exploration time, discrimination indices (differential index, DI) for the training and testing phases are calculated, respectively. Di= (time to search for new object side object-time to search for familiar object side object)/(time to search for new object side object # time to search for familiar object side object) ×100%. The difference between the test stage DI and the training stage DI is a Preference Index (PI) for measuring the spatial memory of the animal.
The results are shown in fig. 6, which shows that ManNAc administration significantly improved spatial memory in hydrocephalus model mice compared to model groups, thereby reducing cognitive impairment resulting from long-term hydrocephalus.
The above results indicate that ManNAc is capable of inhibiting astrocyte proliferation and polarization to neurotoxic forms in acute phase in hydrocephalus model mice; the damage of the white matter myelin sheath of brain tissue is relieved in the chronic period, thereby reducing the ventricular side high signal on magnetic resonance, and finally improving the dyskinesia and long-term cognitive disorder caused by hydrocephalus. Therefore ManNAc was demonstrated to be useful in the preparation of medicaments for the treatment of neurological dysfunction caused by hydrocephalus.
The application of the invention:
in preparing ManNAc into a medicament, an effective amount of ManNAc may be formulated in combination with at least one pharmaceutically acceptable carrier, diluent or excipient. In preparing these compositions, the active ingredient is typically admixed with or diluted with an excipient or enclosed in a carrier which may be in the form of a capsule or sachet. When the excipient acts as a diluent, it may be a solid, semi-solid, or liquid material as a vehicle, carrier, or medium for the active ingredient. Thus, the dosage form may be a liquid, solid or semi-solid dosage form. The liquid preparation can be solution (including true solution and colloid solution), emulsion (including O/W type, W/O type and multiple emulsion), suspension, injection (including water injection, powder injection and transfusion), eye drop, nasal drop, lotion, liniment, etc.; the solid dosage forms can be tablets (including common tablets, enteric coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets), capsules (including hard capsules, soft capsules and enteric coated capsules), granules, powder, micropills, dripping pills, suppositories, films, patches, aerosol (powder) and sprays; the semisolid dosage form may be an ointment, gel, paste, or the like. Examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, and the like. The formulation may further comprise: wetting agents, emulsifying agents, preserving agents (e.g., methyl and propyl hydroxybenzoates), sweetening agents, and the like.
FIG. 1 is a graph showing the results of concentration gradient experiments of ManNAc administration in hydrocephalus model mice;
wherein, the A picture is GFAP/DAPI immunofluorescence double-staining picture, scale 20um. And B is a graph of the base line of the modeling day and the result of the step-by-step experiment at the 7 th day after modeling. Panel C is a graph of statistics of percentage of GFAP+ positive areas around the ventricle.
In the figure, ctr+veh is a control group; hcp+veh as model group; 0.25 represents the 0.25g/kg ManNAc treated group; 0.5 represents the 0.5g/kg ManNAc treated group; 1 represents a 1g/kg ManNAc treatment group; * P <0.05; * P <0.01; * P <0.001.
FIG. 2 is a graph showing the effect of ManNAc on proliferation and polarization of astrocytes in mice in a hydrocephalus model;
wherein, the A graph is GFAP/C3d and GFAP/S100A10 immunofluorescence double-staining graph, and the scale is 20um. And B, a Western Blot detection result diagram of protein expression of periventricular white matter GFAP, C3d and S100A 10. C, D and E are GFAP around ventricle + Percentage of positive area, GFAP + C3d + Cell and GFAP + S100A10 + Cell occupancy GFAP + Cell proportion statistical graph. Panels F, G and H are statistics of the relative GAPDH expression levels of GFAP, C3d and S100A10 protein P around the ventricle;
in the figure, ctr+veh is a control group; hcp+veh as model group; hcp+mannac is a ManNAc-treated group; * P <0.05; * P <0.01; * P <0.001.
FIG. 3 is a graph showing the effect of ManNAc on periventricular white matter demyelination in mice with hydrocephalus models;
wherein, the A graph is a periventricular leucorrhea (CG) and Exocyst (EC) MBP immunofluorescence staining graph, scale 40um. White matter cingulate band (CG) and Exocyst (EC) MBP fluorescence intensity statistics graphs in panels B, C;
in the figure, ctr+veh is a control group; hcp+veh as model group; hcp+mannac is a ManNAc-treated group; * P <0.05; * P <0.01; * P <0.001.
FIG. 4 is a graph showing the effect of ManNAc on the paracentricular elevation signal on MRI in hydrocephalus model mice;
wherein the A diagram is a magnetic resonance T2 weighting sequence skull coronal scan diagram; the diagram B is an Evan's index statistical diagram; c is a statistical graph of the area of the level of the ventricular side elevation signal;
in the figure, ctr+veh is a control group; hcp+veh as model group; hcp+mannac is a ManNAc-treated group; * P <0.001; ns, not signalizing;
FIG. 5 is a graph showing the effect of ManNAc on hydrocephalus model mouse dyskinesia;
wherein the A diagram is the residence time of the rotating rod experiment 7, 14 and 28 days after the molding on the same day; the diagram B is the step-by-step rate of step-by-step experiments 7, 14 and 28 days after the modeling on the same day; panel C is a representative gait footprint of two groups of mice recorded by the Catwalk gait analysis system on day 28 post-molding. Graph D-I is the gait parameters of two groups of mice analyzed by the Catwalk gait analysis system, including step size (graph D), speed (graph E), period of each step (graph F), proportion of standing time in each step (graph G), number of steps per minute (graph H) and diagonal support proportion (graph I);
in the figure, ctr+veh is a control group; hcp+veh as model group; hcp+mannac is a ManNAc-treated group; * P <0.05; * P <0.01; * P <0.001; # #, P <0.001
FIG. 6 is a graph showing the effect of ManNAc on long-term cognitive impairment in hydrocephalus model mice;
wherein the A diagram is a representative motion trail diagram of two groups of mice obtained in a new object recognition experiment testing stage; the diagram B is a difference diagram of preference coefficients (PI) of a new object recognition experiment testing stage and a training stage;
in the figure, ctr+veh is a control group; hcp+veh as model group; hcp+mannac is a ManNAc-treated group; * P <0.05.
While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.
Claims (6)
- Use of n-acetylmannosamine in the manufacture of a medicament for the treatment or prevention of neurological dysfunction caused by hydrocephalus.
- Use of n-acetylmannosamine in the manufacture of a medicament for the treatment or prevention of dyskinesia caused by hydrocephalus, neurodegenerative diseases.
- Use of n-acetylmannosamine in the manufacture of a medicament for improving cognitive disorders.
- The use of n-acetylmannosamine in the preparation of a medicament for inhibiting astrocyte proliferation and promoting astrocyte transformation.
- Use of n-acetylmannosamine in the manufacture of a medicament for reducing white matter demyelinating lesions of brain tissue.
- 6. The use according to any one of claims 1 to 5, wherein the dosage form of the medicament comprises a tablet, powder, granule, capsule, oral liquid, injection or sustained release formulation.
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