Application of HMB in preparation of medicine for improving traumatic brain injury

Abstract

The invention provides application of HMB in preparation of a medicine for improving traumatic brain injury, and belongs to the technical field of biology. As a substance naturally generated in a human body, HMB is extremely high in safety, free of side effects and free of damage to body health. According to the invention, HMB is used for improving cognitive behavior dysfunction after traumatic brain injury for the first time, and a good treatment effect is achieved. In the invention, the HMB can obviously improve nerve injury and cognitive impairment, improve neural behavior defect, spatial memory ability and brain tissue form after traumatic brain injury, relieve encephaledema, reduce expression of GFAP and CD11b at the traumatic brain injury part, inhibit neuroglial cell activation of the traumatic brain injury, and improve the neurological function of the traumatic brain injury. Normal functions of neurons in a TBI state are maintained so as to play a nerve protection role, and the effect of improving cognitive behavior dysfunction after traumatic brain injury is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of HMB in preparing a drug for improving traumatic brain injury. Background Art

[0002] Traumatic brain injury (TBI) is a traumatic structural injury and / or brain dysfunction caused by external forces. It is estimated that there are about 10 million patients with traumatic brain injury every year in the world, of which almost one-third are children and young people. Traumatic brain injury can be roughly divided into two stages: primary brain injury and secondary brain injury. Primary injury is caused by mechanical external force directly acting on the skull and brain, which leads to the death of brain neurons and is an irreversible mechanical injury. Secondary brain injury refers to the subsequent brain tissue damage caused by primary injury, which can last from hours to days or even years. The pathological mechanism of secondary injury is very complex, including neuroinflammatory response, brain edema, destruction of the blood-brain barrier and oxidative stress injury. At the same time, more and more studies have shown that a series of mechanisms that promote cell survival and death, such as microglia, apoptosis, autophagy, and necroptosis, are involved in the development of secondary brain injury. The activation or inhibition of these mechanisms and the balance between them determine the severity and prognosis of secondary brain injury. Therefore, how to find suitable drugs to reduce secondary craniocerebral injury is the key to improving the prognosis of patients with craniocerebral injury. Research on various intervention and regulation mechanisms of secondary brain injury has always been a hot topic in the research of traumatic craniocerebral injury.

[0003] β-Hydroxyβ-methylbutyrate (HMB) is an intermediate metabolite of leucine and was first used in animal husbandry. Today, HMB has been widely used as a muscle supplement by fitness enthusiasts and athletes. At the same time, HMB is not a prescription drug or steroid, but an over-the-counter nutritional supplement that can be easily purchased on the market. Long-term use of HMB is considered safe and has no known side effects.

[0004] Clinically, HMB can also be used as part of the nutritional support treatment for elderly patients with sarcopenia. A domestic randomized controlled clinical study found that after HMB intervention for comatose patients with severe craniocerebral injury, their serum total protein, serum albumin, hemoglobin, muscle strength score, and nutritional status score were significantly improved compared with the conventional enteral nutrition intervention group, and the hs-CRP level was significantly reduced. Therefore, the present invention explores whether HMB as a nutritional supplement can alleviate related complications such as learning and cognitive behavioral disorders caused by TBI. With the deepening of research, HMB is expected to become a new adjuvant therapy for the treatment of TBI. Summary of the invention

[0005] The purpose of the present invention is to provide an application of HMB in the preparation of a drug for improving traumatic brain injury. In the present invention, HMB can significantly improve cognitive behavioral dysfunction after traumatic brain injury.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of HMB in preparing a medicine for improving traumatic brain injury.

[0008] Furthermore, the structure of HMB is shown in formula (I):

[0009]

[0010] Furthermore, the HMB can be used to improve cognitive behavioral dysfunction after traumatic brain injury.

[0011] Furthermore, the HMB can be used to inhibit the activation of glial cells and improve neuronal damage.

[0012] The beneficial effects of the present invention compared with the prior art are:

[0013] As a naturally occurring substance in the human body, HMB is extremely safe, has no side effects, and does not harm physical health. The present invention uses HMB for the first time to improve cognitive behavioral dysfunction after traumatic brain injury, producing a good therapeutic effect. In the present invention, HMB can significantly improve nerve damage and cognitive dysfunction, as well as improve neurobehavioral deficits, spatial memory ability, and brain tissue morphology after traumatic brain injury. In addition, it can also reduce brain edema, reduce the expression of GFAP and CD11b in the site of traumatic brain injury, inhibit the activation of glial cells in traumatic brain injury, maintain the normal function of neurons in the TBI state, and thus play a neuroprotective role, thereby achieving the effect of improving cognitive behavioral dysfunction after traumatic brain injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0015] Figure 1 The effects of TBI and HMB treatment on the water maze test of mice in Example 1, wherein A is a schematic diagram of the swimming trajectory; B is the statistical analysis result of the escape latency; C is the percentage result of the platform quadrant time; D is the result of the number of crossings;

[0016] Figure 2The results of HE staining analysis of changes in brain tissue in the injured area of ​​mice after TBI and HMB treatment in Example 1;

[0017] Figure 3 The basic functions of neurons in the brain tissue of mice in the injured area were analyzed by Nissl staining after TBI and HMB treatment in Example 1;

[0018] Figure 4 This is the quantitative analysis of Nissl bodies in the injury area of ​​mice after TBI and HMB treatment in Example 1;

[0019] Figure 5 The expression of IBA1 protein in the hippocampus of mice after TBI and HMB treatment in Example 1;

[0020] Figure 6 This is a quantitative analysis of the expression of IBA1 protein in mouse hippocampus tissue after TBI and HMB treatment in Example 1;

[0021] Figure 7 The expression of GFAP, a marker of astrocytes in the mouse hippocampus, was analyzed by immunohistochemical staining after TBI and HMB treatment in Example 1;

[0022] Figure 8 This is a quantitative analysis of the expression of GFAP protein in the mouse hippocampus tissue after TBI and HMB treatment in Example 1. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0027] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0028] Example 1

[0029] Example 1 of the present invention detects the damage repair effect of HMB. Each group of experimental mice was subjected to a 4-day Morris water maze positioning and navigation test from the 15th to the 18th day after modeling. The escape latency data of the positioning and navigation test of each group of mice on the 18th day after modeling were statistically analyzed to detect the spatial learning ability of the experimental mice. On the 19th day after modeling, a Morris water maze space exploration test was conducted once, and the number of times the original platform was crossed and the percentage of swimming time in the quadrant of the original platform were statistically analyzed to detect the spatial memory ability of the experimental mice. Each group of mice was sampled at the corresponding time point to observe the gross lesions, HE staining changes and Nissl staining, and the IHC method was used to detect the expression of IBA-1 and GFAP in each group. Specifically, the following steps are included:

[0030] (1) Grouping and modeling

[0031] A. Grouping:

[0032] The experimental mice weighed 25g-30g, and the animal experiments were in accordance with the animal ethics requirements of the Experimental Animal Center of Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, and appropriate anesthetic drugs were used to reduce the pain of the animals during surgery as much as possible. 40 experimental mice that met the standards were selected through the Morris water maze visual platform experiment.

[0033] The experimental mice were fed adaptively in the animal room for one week, and the HMB group and the HMB treatment group were given drinking water containing HMB at a dose of 1g / kg / d. The experimental mice were randomly divided into 4 groups: sham operation (Sham), HMB, model (TBI), and treatment (TBI+HMB). The CCI brain injury model was prepared in the mice of the TBI group and the HMB treatment group, and the mice of the Sham group and the HMB group only had bone windows opened without impact. After modeling, the HMB group and the HMB treatment group continued to be fed with HMB (1g / kg / d) dissolved in drinking water until they were killed for sampling.

[0034] B. TBI model establishment

[0035] The experimental mice were anesthetized with 1.5% isoflurane. After the mice were anesthetized successfully, 1.5% isoflurane was delivered to the mice using an animal anesthesia machine (R500IP, RWD) to maintain anesthesia. After fixing the mouse head, preparing the skin, and disinfecting the scalp with iodine using a brain stereotaxic instrument (SA-100, Yuyan Instrument), the full thickness of the scalp and periosteum were cut longitudinally in the midline to expose the skull. A bone window with a diameter of 4.0 mm was formed in the center of the left parietal bone using an animal skull drill (SD-300, Yuyan Instrument). The TBI model of the experimental mice was established by the controlled cortical impact (CCI) method. TBI was caused in mice using a precision striker (68099H, RWD), and the strike parameters were set as follows: impact velocity (Velocity) = 4.0 m / s, penetration depth (Depth) = 1.5 mm, and duration (Dwell Time) = 0.1 s. After the strike, the mice were in a coma and had a short respiratory arrest. An obvious hematoma appeared immediately in the injured cortex, indicating that the model was successfully prepared.

[0036] After the model was established, the scalp wound was disinfected again with iodine and sutured. The animals in the sham group underwent the same surgical procedure as the animals in the surgery group except for the cortical impact. After the surgery, the animals were placed on a heating pad at 37°C until they fully recovered from anesthesia and were returned to the cage for observation.

[0037] (2) Morris water maze test

[0038] The Morris water maze test was designed by British psychologist Richard G. Morris in 1981. It is mainly used to evaluate the learning, memory and cognitive abilities of animals. It can objectively reflect the changes in the spatial memory, working memory and spatial discrimination abilities of experimental animals. Studies have shown that the Morris water maze test is a powerful and reliable test, and is highly correlated with hippocampal synaptic plasticity and aspartate receptor function. The Morris water maze test can provide more experimental indicators, has reliable sensitivity, and is easy to operate. The Morris water maze test includes the most basic procedures: positioning navigation experiment and spatial exploration experiment, in addition to adaptive training, visual platform experiment and other additional experiments.

[0039] The Morris water maze experimental equipment consists of a circular pool with a black inner wall (diameter 150cm, height 60cm), a circular platform (diameter 10cm, height 22cm), a tracking camera, a computer connected to it and corresponding analysis software. The pool is filled with water, and the water temperature is controlled at 22-24℃. The camera is placed 200cm above the center of the pool to record the position, swimming distance, time and swimming path of the experimental animals.

[0040] The specific contents of the Morris water maze experiment are as follows:

[0041] A. Visual platform experiment: This experiment was conducted before the preparation of the TBI model for 4 days to exclude the influence of factors such as motor and sensory defects on cognitive performance. The platform was placed 2 cm above the water surface. The experimental mice were randomly placed in the water from 4 different quadrants facing the pool wall every day. The interval between each experiment was 15 seconds. The average time for the mice to find the platform (i.e., escape latency) was recorded. The recording stopped 5 seconds after the mice climbed onto the platform. The longest recording time was 90 seconds. If the mice could not climb onto the platform within 90 seconds, they were guided to climb onto the platform and adapt for 10 seconds. After the experiment, the mice were dried and placed in a mouse cage. On the 4th day, qualified mice (mice with escape latency between 20 and 40 seconds) were screened out to prepare the TBI mouse model.

[0042] B. Positioning and navigation experiment: The positioning and navigation experiment was started on the 15th day after TBI modeling, and lasted for 4 days, that is, the 15th, 16th, 17th, and 18th days after TBI modeling, to detect the spatial learning ability of experimental mice. The platform was placed in another quadrant, 2 cm below the water surface, and the mice were randomly placed in the water from 4 different quadrants facing the pool wall every day (the mice were placed on the platform to adapt for 10 seconds before each entry into the water on the first day), and the average time for them to find the platform after 4 entries into the water (i.e., escape latency) was recorded. The recording was terminated 5 seconds after the mouse climbed onto the platform, and the maximum recording time was set to 90 seconds. If the mouse did not climb onto the platform within 90 seconds, it was guided to climb onto the platform to adapt for 10 seconds. After the experiment, the mouse was dried and returned to the cage.

[0043] C. Spatial exploration experiment: This experiment was conducted 24 hours after the last positioning navigation experiment, i.e., 19 days after TBI. It was conducted only once to test the spatial memory ability of the experimental mice. The platform was removed, and the mice were randomly placed in the water from one quadrant facing the pool wall. The number of times the mice crossed the original platform area within 60 seconds, the percentage of swimming time in the quadrant where the original platform was located, and other parameters were recorded. Finally, the mice were dried and returned to the cage. The results were as follows: Figure 1 shown.

[0044] Figure 1 The results of the swimming trajectory diagram in A showed that the search strategies of the mice in the Sham group and the HMB group were mostly linear or trend-based; the search strategies of the mice in the TBI group were mostly random, with a small number of directional; the search strategies of the mice in the HMB treatment group were mostly directional or linear.

[0045] Figure 1 The results of statistical analysis of escape latency in medium B showed that compared with the Sham group, the escape latency of mice in the TBI group was significantly prolonged, and the difference was statistically significant (P<0.05); compared with the TBI group, the escape latency of mice in the HMB treatment group was significantly shortened, and the difference was statistically significant (P<0.05).

[0046] The results of the positioning navigation test showed that after TBI, the escape latency of brain-injured mice in finding the underwater hidden platform was significantly prolonged, and a certain degree of spatial learning dysfunction occurred. After HMB intervention, the escape latency of the mice in the HMB intervention group after TBI was significantly shortened compared with the mice in the TBI group, and the spatial learning dysfunction was improved to a certain extent.

[0047] Figure 1 The results of the percentage of time in the platform quadrant in the middle C showed that compared with the mice in the Sham group, the percentage of time in the platform quadrant of the mice in the TBI group was significantly decreased, and the difference was statistically significant (P<0.05); compared with the mice in the TBI group, the percentage of time in the platform quadrant of the mice in the HMB treatment group was significantly increased, and the difference was statistically significant (P<0.05).

[0048] Figure 1 The results of the number of D crossings showed that compared with the mice in the Sham group, the number of crossings in the TBI group was significantly reduced, and the difference was statistically significant (P<0.05); compared with the mice in the TBI group, the number of crossings in the HMB treatment group was significantly increased, and the difference was statistically significant (P<0.05).

[0049] The results of the spatial exploration test showed that after TBI, the number of times the brain-injured mice crossed the original platform area was significantly reduced, the percentage of swimming time in the original platform quadrant was significantly reduced, and a certain degree of spatial memory dysfunction occurred. Compared with the TBI group mice, the HMB-treated mice crossed the original platform area significantly more times, the percentage of swimming time in the original platform quadrant was significantly increased, and the spatial memory dysfunction was improved to a certain extent.

[0050] (3) HE staining

[0051] A. Preparation of slices:

[0052] Fresh mouse brain tissue samples were fixed in 10% formalin for 24 hours; then dehydrated with gradient ethanol (70%, 80%, 90%, 95%, 100%), with each step of dehydration lasting 1-2 hours; after dehydration, the tissue samples were transparentized twice with xylene, each time for 1 hour; the tissue was immersed in molten paraffin preheated to 56-58°C for 30 minutes to 1 hour to ensure paraffin penetration, and then paraffin was poured into the embedding mold and cooled; 4-5 μm thick sections were cut using a paraffin slicer, and after unfolding in a water bath, they were transferred to a slide coated with an adhesive and dried; thereafter, xylene dewaxing was performed, and the sections were rehydrated with gradient ethanol (100%, 95%, 90%, 80%, 70%), and the quality of the sections was checked under a microscope. After confirmation, they were stored in a dry, dark place for future use.

[0053] B. The sections were immersed in xylene for 2×15min, anhydrous ethanol for 2×5min, and 75% ethanol for 5min, dewaxed and hydrated, and washed with PBS; C. The sections were immersed in hematoxylin solution for 5min, and rinsed with sufficient distilled water for 10min; D. The sections were differentiated with 1% hydrochloric acid alcohol solution for 5s, and washed with distilled water for 5min; E. The sections were treated with hematoxylin bluing solution for 50s, and washed with distilled water for 3min; F. The sections were stained with eosin staining solution for 8min; G. The sections were immersed in anhydrous ethanol for 3×5min and xylene for 2×5min; H. The sections were sealed with neutral gum and observed under a microscope. The results are as follows Figure 2 shown.

[0054] Figure 2The results showed that there was no obvious injury in the brain tissue of the mice in the Sham group, the nerve cells were closely arranged, and the blue-stained nuclei were compact and clearly visible. After TBI, the left injured side of the cerebral cortex of the mice showed obvious injury, and the tissue morphology around the injury was severely damaged, with many intercellular fissures, many ruptured cell bodies, round and enlarged nuclei, and diffuse and lightly stained cytoplasm, involving the cingulate gyrus and hippocampus below the cortex, and an obvious injury notch was seen on the outer side of the upper edge of the hippocampus. The brain tissue morphology of the mice in the HMB treatment group was improved compared with that of the mice in the TBI group, and the cumulative hippocampal area was limited. It can be seen that TBI not only caused extensive damage to the cortical areas related to movement, sensation, reflex, balance and other functions of mice, but also the damage spread to the hippocampus, which is closely related to neurogenesis and spatial learning and memory functions. HMB can improve the neurobehavioral deficits of mice after TBI, reduce brain edema, and improve brain tissue morphology.

[0055] (4) Nissl staining

[0056] A. Paraffin sections were immersed in xylene for 2×15min, anhydrous ethanol for 2×5min, and 75% ethanol for 5min, dewaxed and hydrated, and washed with PBS; B. Nissl stain was used for 5min, and then rinsed with sufficient distilled water; C. 1% hydrochloric acid alcohol solution was used for differentiation for 5s, and then washed with distilled water for 5min; D. Sections were immersed in anhydrous ethanol for 3×5min and xylene for 2×5min; E. The sections were sealed with neutral gum and observed under a microscope. The results are as follows: Figure 3 , 4 shown.

[0057] Figure 3 , 4 The results showed that the number of Nissl bodies in the cortex around the injury focus in the Sham group was large, the arrangement was tight, and the morphology was healthy; the cortical tissue of mice in the TBI model group was loose, and the dark blue tiger-striped Nissl bodies were significantly reduced, reflecting the reduced activity of neurons and severe functional damage; the cortical tissue structure of mice in the HMB treatment group was relatively tight and clear around the injury focus, and the number and morphology were significantly improved compared with those in the TBI group, indicating that HMB helps maintain the normal function of neurons in the TBI state.

[0058] (5) IBA-1 / GFAP immunohistochemistry

[0059] A. Dewax the slices in xylene, anhydrous ethanol, and 75% ethanol in turn until hydrated, and then wash with PBS; B. Immerse the slices in citric acid antigen repair solution (PH 6.0) and microwave at medium heat for 8 minutes, keep warm for 8 minutes, and medium-low heat for 7 minutes. After cooling, wash with PBS for 3×5 minutes; C. Immerse the slices in 3% H2O2 solution, incubate at room temperature in the dark for 25 minutes, and then wash with PBS for 3×5 minutes; D. Use a tissue pen to completely circle the tissue slices, evenly add 3% BSA in the circle, and block at room temperature for 30 minutes; E. Wipe off the blocking solution, add Iba-1 antibody solution (1:500) to the slices, incubate at 4℃ overnight, and then wash with PBS for 3×5 minutes; F. Add secondary antibody (goat anti-rabbit) solution to the slices, incubate at room temperature in the dark for 30 minutes, and then wash with PBS for 3×5 minutes; G. Add 500μL Add 10 μL DAB color source to DAB substrate to prepare DAB colorimetric solution; H. Add DAB colorimetric solution to the slices and incubate for 3-5 minutes until brownish yellow appears, then rinse with tap water to terminate staining; I. Add hematoxylin staining solution to the slices and incubate for 3 minutes, then rinse with tap water for 5 minutes; (10) Differentiate with 1% hydrochloric acid alcohol solution for 5 seconds, then rinse with distilled water for 5 minutes; (11) Treat with hematoxylin bluing solution for 50 seconds, then rinse with distilled water for 3 minutes; (12) Immerse the slices in anhydrous ethanol for 3×5 minutes and xylene for 2×5 minutes in turn; (13) Seal the slices with neutral gum and move them to a microscope for observation.

[0060] IBA-1 antibody can be regarded as a marker protein of microglia in the nervous system. TBI causes a series of stress responses in the brain, microglia are activated in large numbers, IAB-1 staining is significantly enhanced, and cell morphology changes are accompanied. The appearance of brown-yellow granules in the cytoplasm of microglia is a positive expression result.

[0061] Figure 5 , 6 The results showed that most of the microglia in the hippocampus of the Sham group mice were in a resting state, with small and slightly unclear cell bodies, and the overall appearance was roughly a clump-like tree branch morphology; the dark brown marks were obvious in the brain tissue of the TBI group mice, and the number of ameba-like microglia increased, which was manifested by the retraction and thickening of cell processes, the enlargement of cell bodies, and some were in an obvious activated state; compared with the TBI group, the HMB treatment group showed activated microglia, but the ameba morphology was significantly weakened, indicating that HMB inhibited the activation state of microglia in the brain tissue of TBI mice, which can slow down the process of secondary injury of TBI in brain tissue. It can be seen that TBI caused brain damage and triggered a series of oxidative stress reactions, and microglia in the hippocampus were activated from a resting state to an activated state; after HMB intervention, the activation of microglia in the hippocampus was weakened.

[0062] GFAP antibody can be regarded as a marker protein of astrocytes in the nervous system. After brain injury and stress, astrocytes are activated in large numbers, just like microglia, and the sign is a significant increase in the expression of GFAP. The appearance of brown-yellow granules in the cell cytoplasm indicates positive expression. Figure 7 , 8 Show: A small number of GFAP-positive cells were observed in the hippocampus of the brain tissue of mice in the Sham group. Compared with the Sham group, the number of GFAP-positive cells in the TBI group increased significantly. Further observation showed that positive cells were more distributed in the hippocampus. Compared with the TBI group, the number of activated astrocytes in the hippocampus of the HMB-treated group was significantly reduced, indicating that HMB inhibited the activation state of astrocytes in the brain tissue of TBI mice, which can slow down the process of secondary injury of TBI in brain tissue. It can be seen that TBI caused brain damage, which caused extensive oxidative stress response in the TBI group mice, and astrocytes were activated in large numbers in the hippocampus; compared with the TBI group, the activation of astrocytes in the hippocampus of the HMB-treated group was weakened.

[0063] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of HMB in the preparation of drugs for improving traumatic brain injury.

2. The use of HMB in the preparation of a drug for improving traumatic brain injury according to claim 1, characterized in that: The structure of HMB is shown in formula (I):

3. The use of HMB in the preparation of a drug for improving traumatic brain injury according to claim 1, characterized in that: The HMB can be used to improve cognitive behavioral dysfunction after traumatic brain injury.

4. The use of HMB in the preparation of a drug for improving traumatic brain injury according to claim 1, characterized in that: The HMB can be used to inhibit the activation of glial cells and improve neuronal damage.

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