KR20160050858A - Development of Mouse Model for Alzheimer's Disease and Reduction of AD Symptoms through Consumption of Kimchi Lactic Acid Bacteria - Google Patents

Abstract

Alzheimer's disease (AD) is a neurological disorder that causes memory impairment and cognitive decline and is the main cause of nerve damage due to the accumulation of amyloid beta and hyperphosphorylated tau (pTau) proteins. The production of animal models involving these protein changes is an important consideration in the development of AD therapy methods. In this study, it was assumed that changes in exposure to light would affect the brain tissue content, cognitive impairment, and composition of intestinal bacteria in the amyloid beta and pTau protein, and the lactic acid bacteria contained in kimchi soup were supplied to the animal model, . Three groups of mice placed under different light exposure conditions were subjected to two behavioral response tests: grip strength test and object recognition test. As a result, the memory index of the control mice was 1.5 times higher than that of the light restriction group and the light overexposure group, and the amount of amyloid beta and pTau protein analyzed in the brain was higher in the light restriction group. Cognitive function was also reduced when the light was limited. These results indicate that abnormal exposure to light can cause symptoms such as AD and that animals exposed to these conditions can be used as an AD condition model. Next, by classifying the bacteria in the intestines of each mouse, it was proved that the change of the light condition causes the imbalance of bacterial composition in the intestines. After that, Kimchi broth was supplied to both the light restriction group and the light overexposed group, and the same test as object recognition test was performed. The balance of the bacterial composition in the intestine was restored, and the light overexposure group . Amyloid beta and pTau protein contents of the light restriction group were maintained, and a small increase was observed in the other groups. In conclusion, it is possible to construct an AD condition model based on the symptoms of AD in the light restriction group, and the intestinal bacterial composition may affect the cognitive function, the amount of amyloid beta and pTau protein in brain tissue.

Description

[Background Art] [0002] Development of a model of Alzheimer's disease and alleviation of symptoms of Alzheimer's disease by ingesting lactic acid bacteria of Kimchi [

The present invention relates to the development of a model of Alzheimer's disease condition and alleviation of Alzheimer's symptoms through ingestion of Kimchi lactic acid bacteria.

The cause of Alzheimer's dementia has not yet been elucidated, but two of the major causes that are emerging are Amyloid beta and pTau protein. Amyloid beta is produced by cleavage of the precursor amyloid precursor protein (APP) and APP is cleaved by the activities of alpha-secretase, beta- secretase and gamma-secretase. If APP is cleaved by alpha-secretase, Is maintained and is not toxic in the form of sAPP, but amyloid beta is produced when APP is cleaved by beta- secretase and gamma-secretase. The most common forms of amyloid beta are amyloid beta40 and amyloid beta42. When amyloid beta accumulates above a certain concentration, it binds to each other and gradually accumulates as oligomers or plaques. Accumulation of amyloid beta activates microglia and triggers neuritis, leading to degenerative brain disease. As a result, neurotoxicity caused by amyloid beta can damage learning and memory cells, resulting in cognitive deficits in Alzheimer's dementia. Therefore, high concentration of amyloid beta increases the probability of AD production.

pTau is another potential cause of Alzheimer 's dementia. Tau is a protein that helps stabilize microtubules and maintain neuronal structure. However, when abnormally hyperphosphorylated in AD patients, the Tau protein forms a bundle of nerve fibers that no longer functions.

On the other hand, sleeping without good sleep or sleep apnea can cause the accumulation of Ab plaques outside the brain cells and promote AD progression. Human and mouse models have demonstrated the accumulation of amyloid beta as an amyloid beta diurnal pattern, and it has been shown that amyloid beta accumulation can be altered by sleep regulation. (Lucey & Bateman, 2014)

In addition, there are several studies that show that AD is related to bacteria. Infectious burden (IB) caused by bacteria or virus is seriously considered as a pathogenesis of AD. Bu et al. (2014) reported that amyloid beta and inflammatory cytokines were more abundant when infected with four or five pathogenic bacteria than when infected with two pathogens. In other words, IB caused by CMV, HSV-1, B. burgdorferi , C. pneumoniae and H. pylori could be the causative agent of AD. Furthermore, Itzhaki et al. (1997) report that both APOE-e4 (apolipoprotein E e4) carriers and HSV-1 (herpes simplex virus type 1) can cause severe AD. Thus, these studies demonstrate that AD is associated with infection from external causative agents. Conversely, the use of probiotics can control AD, with 10 ¹⁴ microorganisms in the body, most of which are present in intestinal bacteria. Interestingly, these intestinal bacteria control the brain nervous system. (Bhattacharjee et al, 2013)

It is important to develop an ideal AD animal model to confirm the pathogenicity of AD. Currently, genetically modified transgenic animal models are used in AD studies, which take a long time to induce AD pathogenicity. In particular, most of the AD patients are over 65 years old, and the AD transgenic animal model is more similar to the AD that develops early rather than to AD.

Therefore, this study investigates whether the different light conditions are related to the symptoms of Alzheimer 's dementia and to ascertain the possibility of developing an AD condition model in animals exposed to these light conditions changes. In addition, various neurobehavioral tests and amyloid beta and pTau protein analysis were performed to determine whether the supply of Kimchi lactic acid bacteria could induce changes in bacterial composition in the intestine and then reduce AD symptoms.

The present invention provides a method for alleviating symptoms of Alzheimer's disease including the intake of Kimchi lactic acid bacteria, which is characterized by development of an Alzheimer's disease model and alleviation of symptoms of Alzheimer's disease through intake of Kimchi lactic acid bacteria.

Several conclusions were drawn based on the results of various 42-day experiments.

The first conclusion is that the light restriction condition promotes the increase of amyloid beta even during a short period of 4 weeks. In the cognitive ability test, the memory limit of the light restriction group was the lowest. Thus, the light restriction environment means that it is effective to construct the AD condition model for a short period of time.

Light and sleep are related in many ways. If light is present, deep and efficient sleep will be hindered, and related studies support this experimental result. Light can improve sleep pattern and behavior in AD patients, and research has been conducted to demonstrate the correlation between sleep disturbance and AD. Patients with AD often experience changes in sleep-wake cycles, and patients who suffer from sleep problems may progress to AD within two years. (Sterniczuk et al., 2013). Holtzman et al. (2012) also found that sleep disturbance changes the level of amyloid beta around the brain neurons.

Second, when each group is exposed to different amounts of light, the balance in the intestinal bacterial composition may collapse. In mice exposed to environmental changes, such as when people come and go and change light conditions, these changes act as major stresses, leading to an imbalance in intestinal bacterial counts, and imbalances in these intestinal bacteria may lead to impaired cognitive abilities And increased amyloid beta. The supply of Kimchi soup prevented the deterioration of AD symptoms in mice that had already reached a certain level of amyloid beta and pTau before kimchi supply, but when they did not reach a certain level, And pTau protein content were increased.

Kim et al. (2009) reported that dietary fiber, minerals, lactic acid, and beneficial bacteria were found in kimchi, which is a traditional Korean fermented food made of various spices and vegetables (Bang et al, 2008). According to a study by Choi et al. (2014), the object recognition ability is improved by the supply of kimchi soup, and the kimchi extract reduces the action of amyloid beta and prevents brain damage. In addition, Jung et al. (2012) showed that Lactobacillus lactoserobacteria such as pentosus have been shown to prevent memory loss.

Bhattacharjee and Lukiw (2013) analyzed the effect of probiotics on the activity of brain function. The probiotic agent changes the neurochemicals in the brain and the changes improve the neuroimmunity function And that it will be effective in treating and managing AD.

In conclusion, this study has shown that light plays an important role in the production of amyloid beta, a major causative agent of AD, and that sustained exposure to stress increases amyloid beta and pTau protein content. Lactic acid bacteria derived from Kimchi seemed to prevent AD, but could not restore the brain damage that had already occurred before Kimchi was supplied. It will also be possible to produce AD conditional animal models in a short time light restriction environment.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a block diagram illustrating a system for controlling a display apparatus according to an embodiment of the present invention;

Materials and methods

1. Mouse

Sixteen 18-week-old ICR mice were divided into three groups, light restricted group, light overexposed group, and control group, each in six groups. The light restriction group was kept in a dark place for 6 weeks during the experiment and the light over-exposure group was kept in a constantly bright place for 6 weeks. The control group was bright during the day and dark during the night. Body weights of all three groups of mice were measured weekly.

2. Action response test

Four weeks later, two behavioral response tests, grip strength test and object recognition test, were conducted to determine the effects of light exposure on motor function and cognitive function in mice. In the grip strength test, the mice were held upside down on the grate and the time taken for the mice to fall was measured. (Kim, 2006) Object recognition test was used for cognitive function test. Two identical flasks were placed at the opposite ends of the large box and one mouse was placed for 2 minutes to remember the objects. It took about 15 minutes to expose all mice to the same situation. One flask was then replaced with a different shaped object and a 1.5 cm radius was centered around the flask and the new object. Two stopwatches were used to measure the amount of time the mice stayed within a 1.5 cm radius of the old flask and the newly replaced object within a given time. Using the measured values, the memory index was converted as shown in the following equation.

3. Amyloid beta40 and pTau  Measure

Amyloid beta40 and pTau protein concentrations were measured in mouse brain using Elisa Method. Mouse brain tissue was homogenized in 2 ml Ripa buffer containing protease inhibitor and centrifuged at 10,000 rpm for 10 minutes to recover the supernatant. To determine the concentration of amyloid beta40, the human amyloid beta (1-40) Assay Kit (IBL) was used and the assay was performed according to the method provided in the product. The concentration of amyloid beta40 is shown in the graph per mg. pTau measurements were performed using the TAU [pS199] Human ELISA Kit (Invitrogen). The concentration of pTau protein is also shown in the graph per mg.

4. Bacterial culture in the intestine

A. Preparation of Bacterial Medium

Each of the two flasks was charged with 500 ml of distilled water and then added with 35 g of MRS agar (BD Difco ^™ , France) powder to prepare a 70 g / L MRS agar solution. Each flask inlet was covered with aluminum foil, placed in a high-temperature autoclave, and sterilized at 121 ° C and 1.2 atm for 15 minutes. After that, 25 mL of the sterilized solution was immersed in a Petri dish using a pipette, and then poured to make an MRS agar medium. MRS broth (BD Difco ^TM , France) medium was prepared by making a solution of 55 g / L and sterilized before cooling.

B. Mouse Fecal  Collection and culture of intestinal bacteria

Put one mouse in another and wait until you have a bowel movement. Each of the 12 microtubes containing 1 ml of the MRS broth medium was blotted with a vortex mixer. The mixture was looped and streaked onto the MRS agar. One agar plate was inoculated with one mouse feces mixture. Twelve agar plates were placed in a 37 ° C incubator for 24 hours. The cultured intestinal bacteria were observed.

C. Classification of intestinal bacteria

Each bacterial colony was classified according to the characteristics and then cultured separately. Each colony was looped and blended with MRS broth medium, streaked on MRS agar medium, and cultured in a 37 ° C incubator for 24 hours. This sort of culture was repeated 2 or 3 times until one kind of bacteria was cultured on one MRS agar medium. The separated culture broth was sent to Macrogen (Korea) for DNA sequencing of each bacteria and BLAST test . DNA sequencing was performed using the 16s rRNA method, and bacterial species could be identified based on this. The BLAST test was carried out by detecting the DNA sequence of the bacteria and then checking whether they match the DNA sequences of the bacteria already identified.

5. Supply Kimchi soup to mice

A. Isolation of lactic acid bacteria from Kimchi broth

The broth was recovered from kimchi by pipette and mixed with MRS broth medium. The mixture was streaked on the MRS agar plate by streaking method and cultured at 37 ° C for 24 hours. The bacterial colonies were classified according to their characteristics, and then one sort of colony was cultured on one MRS agar plate. Separation culture was repeated 2 or 3 times, and bacteria were identified when only one type of bacterial colony was propagated in one medium.

B. Supply Kimchi broth to mice

The kimchi broth was recovered by purchasing the same type of kimchi as that from which the Kimchi lactic acid bacteria were separated, and mixed with water at a ratio of 1: 2. The diluted kimchi juice was supplied to the light restriction group and the light overexposed group of mice in place of water and replaced with fresh solution every 2 to 3 days. Kimchi soup was supplied for 9 days in total.

C. Observation of changes in mice

To evaluate the changes of Kimchi after feeding Kimchi juice, we performed grip strength test and object recognition test. Mice 1 and 2 in the light overexposed group were euthanized before the experiment, in which 4 mice of the same group were attacked wildly by these mice, causing mice 1 and 2 to be seriously injured to such an extent that they were unable to act. Intestinal bacteria and amyloid beta and pTau protein were also analyzed in all other mice.

D. Classification of bacteria in intestines

Each bacterial colony was classified according to its characteristics. Each colony was mixed with MRS broth medium and inoculated on MRS agar plate and cultured at 37 ° C for 24 hours. Separation culture was repeated until one type of bacterial colony was isolated. DNA sequencing and BLAST test of each bacteria were analyzed.

result

1. Light exposure Amyloid beta Wow pTau  Protein change

To determine whether accumulation of amyloid beta40 and pTau protein, which are the causative agents of AD, in the brain is related to light exposure, we analyzed the brains of light-restricted or overexpressed mice for 4 weeks. Amyloid beta40 was found to accumulate in the light-restricted group of mice, but in the over-exposure group, amyloid beta40 content was similar to that of control mice. Therefore, the restriction of light induces the accumulation of amyloid beta in a short period of time, which can be used to effectively model the condition of Alzheimer's Dementia.

The content of pTau protein in each mouse brain was also measured. The pTau protein content in the mouse brain tissue of the light restriction group and the light overexposure group was higher in all the old groups, which means that abnormal exposure of light induces AD by increasing the level of pTau. The pTau protein of the control group was lower than the other groups.

2. Behavioral responses of mice to light exposure conditions

To determine if light exposure affects the behavioral response of mice, two behavioral response tests were performed: motor function and cognitive function test. The grip strength test, which was performed as a motor function test, measures the dropping time after hanging the mouse on the cage. The mice in the light restriction group had a shorter time to fall, but the difference was so small that they were negligible. Thus, the results of the grip strength test demonstrated that light overexposure did not significantly affect motor function.

For object recognition testing, the mice were placed in two identical flasks to induce learning, and then one of the flasks was replaced with a new object to see if the mouse was interested in the new object. Control mice showed more curiosity with new interest in new objects than most original ones, but mice in light and overexposed groups showed lower memory indices than controls. Memory indexes represent the percentage of time that a mouse is interested in a new object relative to the total time it is interested in the object. Experimental results have shown that light restriction or overexposure can cause serious damage to memory.

As a result, the light restriction group showed a significant increase in Amyloid beta40 and pTau protein content and a decrease in the memory index, indicating that it is possible to produce a condition model of Alzheimer's disease in a short period of time.

3. Changes in intestinal bacteria due to light exposure

Less intestinal bacterial isolates were isolated from the light and overdose groups of mice than from the control. This means that abnormal light exposure is the cause of decreased bacterial diversity in the intestine.

B. tequilensis , B. subtilis , E. faecium , L. plantarum , B. thringiensis and L. sharericus were isolated in the control group . subtilis , E. faecium , E. gallinarum and L. plantarum were isolated. Although the number of bacteria isolated from mice in the light restriction group was less than that of the control group, the number of intestinal bacteria isolated from the whole group of mice was higher than that of the control group. This result indicates that the light restriction limits the different types of bacteria in the intestines for each mouse, which means that light restriction destroys the intestinal bacterial gun balance. Furthermore, E. gallinarum found in the light restriction group is a class of infectious bacteria. (Gilmore, 2002). On the contrary, in the light over-exposure group, not only the number of bacteria per marine was lower than that of the control group, but also the bacterial diversity of the whole group was small. Thus, both overexposure of light and limiting conditions of light were found to be responsible for destroying the balance of bacteria in the intestines.

4. Analysis of Lactic Acid Bacteria Isolated from Kimchi Soup and Changes of Mice after Kimchi Soup Supply

Probiotic agents play a major role in fermented foods, such as studies that mention the potential of probiotics as therapeutic agents for AD. In this study, kimchi which is one of representative traditional foods of Korea was used. As a result, ten kinds of lactic acid bacteria were identified in kimchi, and seven species were genetically identified. The first identified bacteria were Leuconostoc mesenteroides , And is well known for its antitumor function and fibrosarcoma reduction. (Kelkar et al. 1988). The second is Lactobaciullus curieae (Lei et al., 2013). Bacillus sonorensis is widely used as food preservation and remedy. (Chopra et al, 2014). Other Enterococcus faecium is widely used as a probiotic (Kang and Lee, 2005), and Bacillus subtilis is a widely used enzyme for producing various enzymes besides antibiotics and antifungal agents. (Morikawa, 2006) Lactobacillus Sakei are found in fish and meat, and serve to ferment and preserve meat. (Chailou, 2013) This bacterium is often used at the beginning of lactic acid bacteria culture because it prevents the causative bacteria of decay. Bacillus pumilus is known to promote plant growth. (Thomas, 2004)

After the application of kimchi , only three species of bacteria such as E. faecium , L. plantarum and L. sphaericus were isolated in the intestines of the mice. Although the intake of kimchi soup did not increase the intestinal bacterial diversity, it was the result of proving that the eating of the mice greatly influences the composition of intestinal bacteria. This result shows that the intake of food such as kimchi controlled the composition of intestinal bacteria and that probiotics can be used to control AD.

The object recognition test was performed on the same mice and the memory index was measured to see whether it was effective in improving the cognitive ability as well as the change of bacteria in the intestines after the supply of kimchi. In the light over-exposure group, we observed a significant improvement in cognitive ability after the kimchi supply, but the cognitive ability did not improve in the light restriction group. Previously, the amyloid beta and pTau protein contents were high in the light restriction group, so the brain damage would have already proceeded. Because of this, the balance of bacteria in the intestines was restored by the supply of kimchi, but the damaged brain did not show improvement in cognitive function as in the other groups. In contrast, the light overdose group showed a high pTau protein content, but the amyloid beta content was not high, so brain damage was not serious, and kimchi supply seemed to restore some cognitive function.

The mice in each group were placed in a controlled light exposure for 42 days, and during the last 9 days of 42 days, kimchi broth was consumed instead of water. We examined whether kimchi juice intake was effective in decreasing the concentration of amyloid beta and pTau protein. Amyloid beta content was further increased in the control and light overdose groups. This seems to be due to the stress caused by continuous exposure of the mice to the experimenter during the entire period of the experiment. However, the amount of amyloid beta decreased in the light restriction group. Therefore, it is considered that the amyloid beta accumulation is significantly decreased by the kimchi when the amount of amyloid beta is already increased due to the restriction of light.

In Figure 14, the pTau protein in the control group also increased, probably due to increased stress during the experiment. However, the light overexposure group and the light restriction group already had a high level of pTau protein, so kimchi soup seemed to suppress further pTau protein accumulation. On the contrary, when there is a small amount of pTau protein, stress due to the external environment may lead to higher protein accumulation.

Claims (1)

A method for alleviating Alzheimer's symptoms including the ingestion of Kimchi lactic acid bacteria, which comprises developing an Alzheimer's disease model and alleviating Alzheimer's symptoms through ingestion of Kimchi lactic acid bacteria.

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