Application of sialic acid in product for enhancing resistance to external pressure

Abstract

The invention provides an application of sialic acid in a product for enhancing resistance to external pressure, and provides a new application of sialic acid in food, health care products and medicines for promoting resistance to external pressure. Additional sialic acid is supplemented in the early development stage of the mouse, pressure resistance detection is carried out in the puberty, and the result shows that intake of sialic acid in the early life stage can enhance the external environment pressure resistance of the puberty mouse; according to the technical scheme, a new method and feasible reference can be provided for preventing related neurological diseases caused by external pressure.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of sialic acid in products for enhancing resistance to external pressure. Background Art

[0002] Stress is ubiquitous in life, and the main sources of stress come from social, family, physiological, psychological and other factors. When social pressure is too strong or too frequent and exceeds the individual's tolerance, it will lead to emotional disorders such as anxiety and depression, which will seriously affect social life and physical health. Negative emotions or behaviors derived from excessive stress have a negative effect on the development of individuals and society. Therefore, it is urgent to prevent negative behaviors caused by early stress.

[0003] Sialic acid is a nine-carbon sugar derivative. The human body has N-acetylneuraminic acid (Neu5Ac), which is also the main functional component of bird's nest, so it is also called bird's nest acid. The prior art generally believes that sialic acid has anti-inflammatory, antiviral, anti-tumor, and nervous system development-promoting effects, and can be synthesized normally by the human body; but in the early stage of infant liver development is immature, and the demand for sialic acid is large during the critical period of brain development. At this time, the main source of sialic acid is breast milk. Insufficient supplementation of sialic acid in the early stage can lead to defects in bone development and brain development. Sialic acid supplementation outside of breast milk can effectively promote neural development and improve the learning and memory ability of offspring.

[0004] The brain has a complex response mechanism to stress and regulates the individual's emotions and behaviors. In the prior art, sialic acid is used as a neurotrophic supplement to promote neural development and memory. However, the role of sialic acid in other aspects has not been reported, so it is not clear whether sialic acid can enhance the ability to resist external stress. Summary of the invention

[0005] In view of the technical problems existing in the prior art, the present invention discloses an application of sialic acid in a product for enhancing resistance to external pressure.

[0006] To achieve the above-mentioned purpose, the present invention provides an application of sialic acid in a product for enhancing resistance to external pressure, specifically including the role of sialic acid in neuroprotection.

[0007] Preferably, in the product, the sialic acid intake dosage is 10 mg / kg body weight / day.

[0008] Preferably, in the product, the intake mode of sialic acid includes oral administration.

[0009] Preferably, the products include medicines, foods and health products.

[0010] Preferably, the intake period of sialic acid includes intake in early life, which can enhance the ability of adolescence to resist external environmental pressure; wherein, early life can include lactation, infancy to adolescence.

[0011] Furthermore, sialic acid intake early in life could protect against neurological diseases during adolescence.

[0012] Based on the above technical solution, the present invention also provides a drug for enhancing resistance to external pressure, wherein the active ingredient of the drug includes at least sialic acid.

[0013] Preferably, the drug further comprises pharmaceutically acceptable auxiliary ingredients.

[0014] The present invention provides a new use of sialic acid in a product for enhancing resistance to external pressure. Five-day-old mice are used as experimental subjects to construct a mouse model, and after the mice ingest sialic acid, behavioral tests, optical fiber recording experiments and electrophysiological recordings are performed to evaluate the ability of the mice to resist external pressure during adolescence; the behavioral experiments include open field tests, elevated plus maze tests, forced swimming tests, tail suspension tests, etc.

[0015] Specific solutions include:

[0016] (1) Construction of mouse model and sialic acid supplementation

[0017] The mice were raised individually after pregnancy and supplemented with 10 mg / kg body weight of sialic acid every day from the 5th day to the 45th day after the birth of the offspring. The mice in the control group consumed distilled water orally.

[0018] 45 days after birth, the mice underwent behavioral experiments to detect their ability to resist external stress.

[0019] (2) Behavioral experiments

[0020] Open field test: The open field can provide a low-stress, empty environment and detect the behavioral responses of mice in a low-stress environment.

[0021] The size of the open field used in the behavioral experiment of the present invention is 50cm*50cm*50cm. A camera is placed above the open field and the movement of mice is recorded by Anymaze behavioral software. At the beginning of the experiment, the mice are gently placed in the center area of ​​the open field, and the movement time of the mice in the 25cm*25cm area in the center of the open field and the number of times they enter and exit the center area in the next 5 minutes are used as indicators to measure the mice's ability to resist stress.

[0022] Elevated plus maze: The elevated plus maze is divided into open arms and closed arms. The whole device is 50 cm above the ground. The size of the open arms and closed arms is 30 cm*8 cm. The closed arms are surrounded by 15 cm high opaque boards to create a closed environment, and the open arms are unobstructed to create a low-pressure environment. At the beginning of the experiment, the mice were gently placed in the central area of ​​the elevated plus maze (8 cm*8 cm) facing the open arm. The time the mice moved in the open arm area and the number of times they entered and exited the central area in the next 5 minutes were used as indicators to measure the mice's ability to resist stress.

[0023] Forced swimming: By placing the experimental animals in a confined environment (such as water), the animals struggle desperately to escape but cannot escape, thus providing an unavoidable oppressive environment. After a period of experiment, the animals show a typical "immobility state", reflecting a state called "behavioral despair". The diameter of the forced swimming device is 20 cm and the water depth is 20 cm. The immobility time of the mice within 5 minutes after being placed in the water is used to judge the ability of the mice to resist the inevitable pressure.

[0024] Tail suspension test: The tail of the experimental animal is fixed so that its head is suspended downward. The animal struggles in this environment and tries desperately to escape but cannot escape, thus providing an unavoidable oppressive environment. After being unable to escape after efforts, it becomes intermittently immobile, showing a state of "behavioral despair". During the experiment, the mouse is more than 40 cm above the ground, and the immobility time within 5 minutes after the tail suspension is used to judge the mouse's ability to resist inevitable pressure.

[0025] (3) Fiber Optic Recording Experiment

[0026] The medial prefrontal cortex (mPFC) is a key brain region involved in stress response. The present invention uses the virus AAV-CaMKII-GCaMP6s that can convert neuronal calcium activity into light signals to reflect the mPFC response of mice when facing a stressful environment. After 38 days of birth, mice were anesthetized with 60mg / kg sodium pentobarbital and fixed on a stereotaxic injection fixture. After cutting the scalp, leveling, marking the injection site (AP 1.8mm, ML 0.5mm, DV 2.0mm), drilling and completing the injection operation, the injection needle was slowly lifted after the injection was completed and the needle was stopped for 10 minutes, and then a ceramic optical fiber was buried 0.1mm above the injection site, fixed with dental cement and skull nails, and the optical fiber recording experiment was performed 2 weeks later.

[0027] The optical fiber recording experiment was performed when the mice were suspended by their tails. The 480nm wavelength signal was used as the target signal, and the 405nm wavelength signal was used as the environmental noise interference. The 480nm signal was deducted from the 405nm signal to record the corrected signal. The data analysis was performed using the inper exclusive data processing program. The time points from the static state to the struggle of the mice during tail suspension were used as the marking points, and the mPFC neuronal activity before and after the marking points was analyzed. The collected fluorescence signal data was presented in the form of dfff, and the amplitude of the peak generated during the struggle and the time to reach the peak reflected the neuronal activity of the mice.

[0028] (4) Electrophysiological recording

[0029] The mice were quickly anesthetized with isoflurane, then cut off and the brain was removed. The slices were cut in a slicing solution ice-water mixture with continuous flow of 95% O2 / 5% CO2. The slicing solution ice-water mixture included 22.5mM KCl, 7mM MgSO4·7H2O, 0.5mM CaCl2·2H2O, 1.25mM NaH2PO4·H2O, 25mM NaHCO3, 10mM glucose (Glucose), 210mM ​​sucrose (Sucrose), saturated with 95% O2 / 5% CO2, pH = 7.3-7.4.

[0030] After the brain slice (300um) was cut, it was transferred to oxygen-saturated artificial cerebrospinal fluid (ACSF) and incubated at 34°C for half an hour, and then transferred to 27°C and incubated for at least 1 hour. The artificial cerebrospinal fluid included 119mM NaCl, 2.5mM KCl, 1mMNaH2PO4·H2O, 26.2mM NaHCO3, 2.5mM CaCl2·2H2O, 1.3mM MgCl2·6H2O, 11mM Glucose, saturated with 95% O2 / 5% CO2, pH = 7.3-7.4, osmolarity = 290-300mOsm.

[0031] The brain slices were placed in continuously oxygenated ACSF, and the target brain area was found under a microscope before recording. The glass electrodes used for recording were pulled by an electrode puller to a resistance of 3-6 MΩ; when recording microexcitatory postsynaptic current (mEPSC), the voltage clamp mode was used, and the clamping potential was -70 mv; when recording action potential (AP), the current clamp mode was used, and the potential was not clamped.

[0032] The recording electrode solution included 110 mM K-gluconate (gluconate), 40 mM KCl, 10 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 3 mM Mg-ATP, 0.5 mM Na-GTP and 0.2 mM EGTA (ethylene glycol ditetraacetic acid), pH = 7.3-7.4, osmotic pressure = 290-300 mOsm; TTX (1 μM) and PTX (250 μM) were added to artificial cerebrospinal fluid (ACSF) to isolate miniature excitatory postsynaptic currents (mEPSCs).

[0033] The present invention uses the frequency and amplitude of the collected conductive mEPSC signal to reflect the change of the synaptic transmission function of the neuron, and uses the frequency and threshold of AP release to reflect the neuronal activity.

[0034] The technical effects of the technical solution of the present invention are as follows:

[0035] By giving mice additional sialic acid supplements in the early stages of their development and testing their stress resistance during adolescence, it was found that sialic acid supplementation in early life can enhance the ability of adolescent mice to resist external environmental pressure. This technical solution provides a new method and feasibility reference for preventing the occurrence of related neurological diseases caused by external pressure, provides new ideas for nutritional supplements from infants to adolescence, broadens the prospects for the application of food and health care products resources, and provides a new solution for the development of drugs to enhance external pressure. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a comparison diagram of open field movement trajectories of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0037] Figure 2 The figure is a comparison of the movement time in the central area of ​​the open field between mice that ingested sialic acid and mice in the control group according to a typical embodiment of the present invention.

[0038] Figure 3 This is a comparison chart of the number of times that mice that ingested sialic acid and mice in the control group entered the center of the open field in a typical embodiment of the present invention.

[0039] Figure 4 This is a comparison diagram of the elevated plus maze movement trajectories of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0040] Figure 5 The figure is a comparison of the open arm movement time of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0041] Figure 6 The figure is a comparison chart of the number of times that mice that ingested sialic acid entered the open arms and mice in the control group according to a typical embodiment of the present invention.

[0042] Figure 7 It is a schematic diagram of a mouse forced swimming test in a typical embodiment of the present invention and a comparison diagram of the immobility time in the forced swimming test between mice that ingested sialic acid and mice in the control group.

[0043] Figure 8 It is a schematic diagram of a mouse tail suspension test in a typical embodiment of the present invention and a comparison diagram of the immobility time of the mouse tail suspension test between mice that ingested sialic acid and mice in the control group.

[0044] Fig. 9 Schematic diagram of mouse virus injection and optical fiber embedding in a typical implementation scheme of the present invention.

[0045] Fig.10 This is a schematic diagram of a mouse brain slice with optical fiber implantation sites in a typical embodiment of the present invention.

[0046] Fig.11 Schematic diagram of the operation flow of a mouse optical fiber recording experiment in a typical implementation scheme of the present invention.

[0047] Fig.12 This is a representative trajectory diagram of the excitatory neuron activities in the medial prefrontal cortex during optical fiber recording of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0048] Fig.13 The figure shows the average changes in neuronal activity when mice that ingested sialic acid and control mice struggled in a typical embodiment of the present invention.

[0049] Fig.14 This is the average peak value of neuronal activity during the struggling phase of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0050] Fig.15 The time from the start of struggling to the peak of neuronal activity for mice that ingested sialic acid and control mice in a typical embodiment of the present invention.

[0051] Fig.16 This is a heat map of neuronal activity during the struggle of mice that ingested sialic acid and control mice in a typical embodiment of the present invention.

[0052] Fig.17a Graph showing representative waveforms of mEPSC of medial prefrontal pyramidal neurons in mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0053] Fig.17b This is a comparison chart of the mEPSC firing frequency between mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention.

[0054] Fig.17cThis is a comparison diagram of the mEPSC signal amplitudes of mice that ingested sialic acid and mice in the control group in a typical embodiment of the present invention. DETAILED DESCRIPTION

[0055] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the protection scope of the present invention.

[0056] The technical scheme of the present invention is further described in detail below through specific examples. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0057] Example 1

[0058] 1. Mouse Model Construction and Sialic Acid Uptake

[0059] The mice were raised individually after pregnancy and supplemented with 10 mg / kg body weight of sialic acid every day from the 5th day to the 45th day after the birth of the offspring. The mice in the control group consumed distilled water orally.

[0060] 45 days after birth, the mice underwent behavioral experiments to detect their ability to resist external stress.

[0061] 2. Stereotaxic Injection and Fiber Optic Implantation

[0062] The medial prefrontal cortex (mPFC) is a key brain region involved in stress response. The present invention uses AAV-CaMKII-GCaMP6s, a virus that can convert neuronal calcium activity into light signals, to react to the mPFC response of mice when facing a stressful environment. After 60mg / kg sodium pentobarbital deep anesthesia, mice 38 days after birth are fixed on a stereotaxic injection fixture to ensure that the brain position will not move in the subsequent operation. The scalp is cut along the suture position of the top of the head with sterilized surgical scissors to completely expose the skull and the anterior and posterior fontanelles. With the anterior fontanelle as the three-dimensional origin, the brain is roughly leveled with the naked eye, and then the stereotaxic instrument and stereoscope are used for detailed leveling to ensure that the anterior and posterior fontanelles are in the same horizontal plane and the left and right skulls are level. After leveling, a stereotaxic instrument and an injection needle are used to mark the virus injection site, and a skull drill is used to carefully drill a hole with an aperture of about 1mm. The operation process is to avoid excessive force and bleeding as much as possible.

[0063] After probing and ensuring that the injection needle can penetrate into the injection site, perform virus aspiration and injection. Virus aspiration is performed at a speed of 100nL / second, and virus injection is performed at 30nL / minute. After the virus injection is completed, stop the needle for 10 minutes to avoid virus leakage, and then slowly lift the virus injection needle until it is completely withdrawn. Use a fiber optic clamp to bury the optical fiber. After the optical fiber is fixed on the fiber optic clamp, use the bregma as the origin of the three-dimensional coordinates, insert the tip of the optical fiber to 0.1mm above the virus injection site, and insert the optical fiber as slowly as possible. After the optical fiber is inserted, use dental cement and skull nails to fix it to prevent the optical fiber from falling off. After the dental cement hardens, remove the fixator from the mouse and place it on a constant temperature heating pad to wait for recovery.

[0064] After cutting the scalp, leveling, marking the injection site (sites include AP 1.8mm, ML 0.5mm, DV 2.0mm), drilling and completing the injection operation, the injection needle was slowly lifted after the injection was completed and the needle was stopped for 10 minutes. Then, a ceramic optical fiber was buried 0.1mm above the injection site and fixed with dental cement and skull nails. The optical fiber recording experiment was carried out 2 weeks later.

[0065] See also Fig. 9 Schematic diagram of virus injection and optical fiber embedding in mice. Fig.10 This is a schematic diagram of the brain slice where the optical fiber is buried. Fig.11 Schematic diagram of the operation flow of mouse optical fiber recording experiment.

[0066] 3. Acute Brain Slice Preparation for Electrophysiology

[0067] Before preparing acute brain slices of mice, pre-cool the surgical instruments and high-glucose slicing solution. After freezing the high-glucose slicing solution into an ice-water mixture, continue to ventilate for at least 20 minutes. Preheat the artificial cerebrospinal fluid in a 33°C water bath and pass 95% oxygen / 5% carbon dioxide to achieve oxygen saturation. Remove the surgical instruments, fill the slice slot of the slicer with ice, and then anesthetize the mouse to prepare for brain slicing.

[0068] After the mouse was rapidly anesthetized with isoflurane, it was immediately decapitated. The brain was removed on an ice dish. The mouse brain tissue was removed within 20 seconds and placed in a mixture of ice and water in a high-glucose slicing solution to cool for 1 minute. After cooling, the brain tissue was removed and trimmed with a blade, glued to the slicer tray with glue, the tray was installed and poured with a mixture of ice and water in a high-glucose slicing solution, the blade was adjusted, and the target tissue (300 microns) was gradually trimmed and cut. The brain slice was transferred to preheated 34°C artificial cerebrospinal fluid using a Pasteur pipette, recovered for 30 minutes, and then transferred to 27°C for incubation for at least 1 hour, and finally the patch clamp recording operation was performed.

[0069] Experimental test:

[0070] 1. Behavioral Experiment

[0071] (1) Open field test: The open field test can provide a low-stress environment and detect the behavioral responses of mice in a low-stress environment.

[0072] The size of the open field used in the behavioral experiment of this example was 50 cm*50 cm*50 cm. A camera was placed above the open field and the movement of mice was recorded using Anymaze behavioral software.

[0073] At the beginning of the experiment, the mice were gently placed in the center area of ​​the open field. The time the mice moved in the 25cm*25cm area in the center of the open field and the number of times they entered and exited the center area in the next 5 minutes were used as indicators to measure the mice's ability to resist stress.

[0074] See also Figure 1-Figure 3 , is a diagram of the open field movement trajectory of mice in the open field experiment in which sialic acid promotes the ability of mice to resist low-stress environment. By comparing with the mice in the control group, the number of times the mice that ingested sialic acid crossed the central area of ​​the open field and the movement time in the central area increased significantly, indicating that the ability of mice to resist low-stress open environment has been enhanced.

[0075] (2) Elevated plus maze: The elevated plus maze consists of an open arm and a closed arm. The entire device is 50 cm above the ground. The dimensions of the open arm and the closed arm are both 30 cm*8 cm. The closed arm is surrounded by an opaque board 15 cm high to create a closed environment, and the open arm is unobstructed to create a low-stress environment. At the beginning of the experiment, the mouse was gently placed in the central area (8 cm*8 cm) of the elevated plus maze facing the open arm. The time the mouse spent in the open arm area and the number of times it entered and exited the central area in the next 5 minutes were used as indicators to measure the mouse's ability to resist stress.

[0076] See also Figure 4-Figure 6 This is a comparison of the elevated plus maze of mice that ingested sialic acid and mice in the control group. While there was no difference in the number of times they entered and exited the open arms, the mice that ingested sialic acid stayed in the open arms more time, indicating that the mice's ability to resist low-stress environments was enhanced.

[0077] (3) Forced swimming test (FST): By placing the experimental animals in a confined environment (such as water), the animals struggle desperately to escape but cannot escape, thus providing an unavoidable oppressive environment. After a period of experiment, the animals show a typical "immobility state", reflecting a state called "behavioral despair". The diameter of the forced swimming apparatus is 20 cm and the water depth is 20 cm. The immobility time of the mice within 5 minutes after being placed in the water is used to judge the ability of the mice to resist inevitable pressure.

[0078] Figure 7It is a simulation diagram of the forced swimming experiment on mice and a comparison diagram of the forced swimming experiment between mice that ingested sialic acid and mice in the control group. It can be seen from the figure that the mice that ingested sialic acid stayed immobile in the water for a shorter time, that is, the time of behavioral despair was shorter, indicating that the ability of mice to resist inevitable stress was enhanced.

[0079] (4) Tail suspension test (TST): The tail of the experimental animal is fixed so that its head is suspended downward. The animal struggles in this environment and tries desperately to escape but cannot escape, thus providing an unavoidable oppressive environment. After being unable to escape after efforts, the animal becomes intermittently immobile, indicating a state of "behavioral despair". During the experiment, the mouse is more than 40 cm above the ground, and the immobility time within 5 minutes after the tail suspension is used to judge the mouse's ability to resist inevitable pressure.

[0080] Figure 8 It is a simulation diagram of the tail suspension experiment on mice and a comparison diagram of the tail suspension experiment between mice that ingested sialic acid and mice in the control group. It can be seen from the figure that the mice that ingested sialic acid had a shorter immobility time in the tail suspension state, that is, the behavioral despair time was shorter, indicating that the mice's ability to resist inevitable stress was enhanced.

[0081] (5) Fiber Optic Recording Experiment

[0082] The optical fiber recording experiment was performed when the mice were subjected to the tail suspension experiment. The 480nm wavelength signal was used as the target signal, and the 405nm wavelength signal was used as the environmental noise interference. The 480nm signal was deducted from the 405nm signal to record the signal after correction. The data analysis was performed using the inper exclusive data processing program. The time points from the static state to the struggle of the mice during tail suspension were used as the marking points, and the mPFC neuronal activity before and after the marking points was analyzed. The collected fluorescence signal data was presented in the form of df / f, and the amplitude of the peak generated during the struggle and the time to reach the peak reflected the neuronal activity of the mice.

[0083] Fig.12 This is a representative trajectory of excitatory neuronal activity in the medial prefrontal cortex of mice during optical fiber recording. Fig.13 The average change in neuronal activity when the mice struggled, Fig.14 is the average peak value of neuronal activity during the mouse struggling stage. Fig.15 It is the time from when the mouse starts struggling to when the neuronal activity reaches the peak; Fig.16This is a heat map of neuronal activity when mice struggle; as can be seen from the figure, compared with the control group, when the mice that ingested sialic acid resisted inescapable pressure, the mPFC brain region neuronal activity was more active throughout the resistance phase, and the neuronal response corresponding to a single struggle was stronger, with a higher peak response. The above results show that there is a strong correlation between the resistance of mice and the mPFC brain region neuronal activity, and the mPFC neuronal response of mice that ingested sialic acid was stronger.

[0084] (6) Electrophysiological recording

[0085] The mice were quickly anesthetized with isoflurane, then cut off and the brain was removed. The slices were cut in a slicing solution ice-water mixture with continuous flow of 95% O2 / 5% CO2. The slicing solution ice-water mixture included 22.5mM KCl, 7mM MgSO4·7H2O, 0.5mM CaCl2·2H2O, 1.25mM NaH2PO4·H2O, 25mM NaHCO3, 10mM glucose (Glucose), 210mM ​​sucrose (Sucrose), saturated with 95% O2 / 5% CO2, pH = 7.3-7.4.

[0086] After the brain slice (300um) was cut, it was transferred to oxygen-saturated artificial cerebrospinal fluid (ACSF) and incubated at 34°C for half an hour, and then transferred to 27°C and incubated for at least 1 hour. The artificial cerebrospinal fluid included 119mM NaCl, 2.5mM KCl, 1mMNaH2PO4·H2O, 26.2mM NaHCO3, 2.5mM CaCl2·2H2O, 1.3mM MgCl2·6H2O, 11mM Glucose, saturated with 95% O2 / 5% CO2, pH = 7.3-7.4, osmolarity = 290-300mOsm.

[0087] The brain slices were placed in continuously oxygenated ACSF, and the target brain area was found under a microscope before recording. The glass electrodes used for recording were pulled by an electrode puller to a resistance of 3 to 6 MΩ. When recording microexcitatory postsynaptic currents (mEPSCs), the voltage clamp mode was used, and the clamping potential was -70 mv; when recording action potentials (APs), the current clamp mode was used, and the potential was not clamped. The recording electrode solution included 110 mM K-gluconate (gluconate), 40 mM KCl, 10 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid), 3 mM Mg-ATP, 0.5 mM Na-GTP and 0.2 mM EGTA (ethylene glycol ditetraacetic acid), pH = 7.3-7.4, osmotic pressure = 290-300 mOsm; TTX (tetrodotoxin, 1 μM) and PTX (paclitaxel, 250 μM) were added to artificial cerebrospinal fluid (ACSF) to isolate miniature excitatory postsynaptic currents (mEPSCs).

[0088] Fig.17a This is a representative waveform of mEPSC in mouse medial prefrontal pyramidal neurons; Fig.17b is the frequency of mEPSC firing; Fig.17c is the mEPSC signal amplitude; as can be seen from the figure, compared with the control group, the frequency and amplitude of mEPSC in the mPFC neurons of mice that ingested sialic acid increased significantly, indicating that the synaptic transmission activity of the mPFC neurons in mice was enhanced after ingesting sialic acid.

[0089] Perform data analysis on the results of the above experiments:

[0090] All values ​​are expressed as mean ± standard error (SEM). The data were statistically analyzed using independent sample t test and one-way analysis of variance, and the results were presented in bar graphs. P < 0.05 was considered statistically significant.

[0091] Experimental results analysis

[0092] Combined with the above experimental results, it is shown that supplementing sialic acid in early life can enhance the ability of adolescent mice to resist external environmental pressure, and when resisting external environmental pressure, the real-time activity of neurons in the mPFC brain region of mice that ingested sialic acid was significantly enhanced. This enhancement in real-time neuronal activity may be due to the enhanced synaptic transmission activity between neurons, which makes the mPFC neurons respond more strongly, ultimately reflecting the improvement in the ability to resist external environmental pressure in behavior.

[0093] Furthermore, the above experimental results show that giving mice additional sialic acid supplements in the early stages of development and testing their stress resistance during adolescence show that sialic acid supplementation in early life can enhance the ability of adolescent mice to resist external environmental stress.

[0094] The above are only preferred embodiments of the present invention, which do not limit the protection scope of the present invention. For those skilled in the art, the present invention may have various modifications and changes. Within the spirit and principle of the present invention, changes, modifications, replacements, integrations and parameter changes to these embodiments by conventional substitutions or by being able to achieve the same functions without departing from the principles and spirit of the present invention all fall within the protection scope of the present invention.

Claims

1. A use of sialic acid in a product for enhancing resistance to external stress, specifically including the role of sialic acid in neuroprotection.

2. The use according to claim 1, characterized in that: In the product, the dosage of sialic acid intake is 10 mg / kg body weight / day.

3. The use according to claim 2, characterized in that: In the product, the intake method of sialic acid includes oral administration.

4. The use according to claim 1, characterized in that: The products include medicines, food and health products.

5. The use according to any one of claims 1 to 4, characterized in that: The intake period of sialic acid includes intake in early life to enhance resistance to external environmental stress during adolescence.

6. The application according to claim 5, characterized in that: Sialic acid intake early in life may protect against neurological diseases during adolescence.

7. The use according to claim 5, characterized in that: The early life period includes the lactation period, infancy to adolescence.

8. A food for enhancing resistance to external stress, comprising at least sialic acid.

9. A drug for enhancing resistance to external stress, comprising at least sialic acid.

10. The drug for enhancing resistance to external pressure according to claim 9, characterized in that: The medicament further comprises pharmaceutically acceptable auxiliary ingredients.

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