阅读说明：本技术 衣康酸二甲酯在制备治疗和/或预防神经退行性疾病的药物中的应用 (Application of dimethyl itaconate in preparation of medicine for treating and/or preventing neurodegenerative diseases ) 是由 潘伟 于英华 赵进秀 何妍 颜子易 徐大祥 杨晓莹 张鹏 胡敏敏 胡涛 李梦迪 于 2021-10-08 设计创作，主要内容包括：本发明属于退行性疾病治疗药物技术领域,涉及衣康酸二甲酯在制备治疗和/或预防神经退行性疾病的药物中的应用。经过试验验证,衣康酸二甲酯能够减轻学习和记忆能力下降、突触超微结构损伤、降低神经炎症。(The invention belongs to the technical field of degenerative disease treatment medicines, and relates to application of dimethyl itaconate in preparation of medicines for treating and/or preventing neurodegenerative diseases. Experiments prove that the dimethyl itaconate can relieve the decline of learning and memory abilities, the damage of synaptic ultrastructure and reduce neuroinflammation.)

1. Application of dimethyl itaconate in preparing medicine for treating and/or preventing neurodegenerative diseases.

2. The use of claim 1, wherein the dimethyl itaconate prevents neuroinflammatory responses by inhibiting TNF-a and IL-6 secretion from microglia.

3. The use according to claim 1, wherein dimethyl itaconate is used in the manufacture of a medicament for ameliorating obesity-induced cognitive function impairment.

4. The use according to claim 3, wherein the cognitive impairment is:

impairment of cognitive function characterized by decreased learning, memory and self-care ability, and/or

Impairment of cognitive function characterized by impairment of synaptic ultrastructures, and/or

Impairment of cognitive function characterized by neuroinflammation.

5. The use according to claim 3, wherein the dimethyl itaconate is useful in the preparation of a medicament for ameliorating the decline in learning, memory and self-care of life caused by obesity.

6. The use of claim 3, wherein the dimethyl itaconate prevents synaptic ultrastructural damage by increasing the thickness of post-synaptic membrane protein PSD95, increasing the active band length, decreasing the synaptic gap width.

7. The use according to claim 3, wherein the dimethyl itaconate is useful for the preparation of up-regulators of the expression of presynaptic membrane protein SYN, postsynaptic membrane protein PSD95 and neurotrophic factor BDNF.

8. The use according to claim 3, characterized in that the dimethyl itaconate can be used for the preparation of inhibitors of the proliferation of microglia in hippocampus CA1, CA3, DG; and/or

The dimethyl itaconate can be used for preparing expression down-regulators of hippocampal mRNA levels of CD68, TNF-alpha, IL-6 and IL-1 beta.

9. Use according to claim 1, wherein the neurodegenerative disease is alzheimer's disease or obesity-related cognitive disorders.

10. A medicament for the treatment and/or prevention of neurodegenerative diseases, characterized in that it comprises as an active ingredient dimethyl itaconate according to any one of claims 1 to 9, in association with a pharmaceutically acceptable adjuvant or carrier.

Technical Field

The invention belongs to the technical field of degenerative disease treatment medicines, and relates to application of dimethyl itaconate in preparation of medicines for treating and/or preventing neurodegenerative diseases.

Background

Alzheimer's Disease (AD), the major form of senile dementia, is a major medical problem facing worldwide in common. 2018 the international association for alzheimer's disease published reports showing that 1 AD patient is produced every 3 seconds worldwide. It is estimated that by 2030, the global AD associated economic cost is $ 2 trillion. To date, there is a lack of effective drugs and therapies for the treatment of AD. Because the pathogenesis of the disease is not completely clear, most of patients are in the late stage of the disease when being clinically diagnosed, and the neuropathy in the brain is difficult to reverse through medicines.

Obesity is an epidemic, systemic metabolic disease that seriously harms human health. 2016, reports that about 6 hundred million obese people are around the world, and the number of obese people in China exceeds the first global position of the American ranks. The epidemiological survey of people and animal experiments show that obesity not only increases the risk of diseases such as hypertension, diabetes, cancer and the like, but also can cause the decline of cognitive function, and is an important cause for inducing cognitive dysfunction and Alzheimer disease. Therefore, exploring the cognitive impairment intervention strategy associated with obesity would be helpful in preventing and treating AD.

Cognitive dysfunction is a major feature of AD. In recent years, studies have found that neuroinflammation plays an important role in promoting cognitive impairment. On one hand, microglia serving as an inherent immune cell of a central nervous system is widely activated in a neuroinflammation environment, and can phagocytose synapses, damage synapse ultrastructures and cause cognitive damage; on the other hand, these immune cells can also produce large amounts of pro-inflammatory cytokines, mediating disease progression. According to earlier researches, a large amount of microglia in the brain area of an obese mouse are activated to induce neuroinflammation, damage a synaptic ultrastructure and cause behavioral abnormalities related to learning, memory and life self-care, so that the neuroinflammation is an important basis of cognitive injury caused by obesity. Therefore, improvement of neuroinflammation and repair of neuronal damage are important strategies for treatment of cognitive dysfunction.

Itaconic acid is a metabolite produced in the tricarboxylic acid cycle and catalyzes the decarboxylation of aconitic acid by aconitate decarboxylase 1. It is well known that itaconic acid has strong bactericidal and antiviral effects. The rise of immune metabolism greatly widens the understanding of the role of itaconic acid in immune regulation mechanism and diseases. In macrophages stimulated by LPS in vitro, it is proved that itaconic acid is an important connecting point of macrophage immunity and metabolism and is a key metabolic node for negatively regulating macrophage inflammatory response. Thus, itaconic acid has potential for the control of inflammatory diseases. However, since natural itaconic acid cannot pass through cell membranes, development and application thereof are limited. The itaconic acid derivative-Dimethyl Itaconate (DI) can smoothly pass through cell membranes, and has good anti-inflammatory effect. However, it has not been reported whether dimethyl itaconate can improve cognitive function impairment and neurodegenerative diseases caused by obesity.

Disclosure of Invention

In order to solve the technical problems, the invention provides the following technical scheme:

the invention provides an application of dimethyl itaconate in preparing a medicament for treating and/or preventing neurodegenerative diseases.

Preferably, the dimethyl itaconate prevents neuroinflammatory reaction by inhibiting secretion of TNF-alpha and IL-6 by microglia.

Preferably, the dimethyl itaconate is used for the preparation of a medicament for ameliorating obesity-induced cognitive function impairment.

Preferably, the cognitive function impairment is:

cognitive impairment characterized by a decline in learning, memory and self-care ability; and/or

Cognitive function impairment characterized by synaptic ultrastructural impairment; and/or

Impairment of cognitive function characterized by neuroinflammation.

Preferably, the dimethyl itaconate can be used for preparing a medicament for improving the decline of learning, memory and self-care ability caused by obesity.

Preferably, the dimethyl itaconate prevents synaptic ultrastructural damage by increasing the thickness of postsynaptic membrane protein PSD95, increasing the length of the active band, and decreasing the width of synaptic cleft.

Preferably, the dimethyl itaconate can be used for preparing an expression up-regulator of presynaptic membrane protein SYN, postsynaptic membrane protein PSD95 and neurotrophic factor BDNF.

Preferably, the dimethyl itaconate can be used for preparing a proliferation inhibitor of microglia in hippocampus CA1, CA3 and DG; and/or

The dimethyl itaconate can be used for preparing expression down-regulators of hippocampal mRNA levels of CD68, TNF-alpha, IL-6 and IL-1 beta.

Preferably, the neurodegenerative disease is alzheimer's disease or obesity-related cognitive disorders.

The invention also provides a medicament for treating and/or preventing neurodegenerative diseases, which takes the dimethyl itaconate as an active ingredient and is supplemented with pharmaceutically acceptable auxiliary materials or carriers.

Compared with the prior art, the invention has the beneficial effects that:

1. the invention provides an application of dimethyl itaconate in preparing a medicament for treating and preventing neurodegenerative diseases, wherein the dimethyl itaconate can prevent neuroinflammatory reaction by inhibiting microglia from secreting TNF-alpha and IL-6; also can treat and/or prevent cognitive function impairment diseases induced by obesity.

2. The experimental study of the invention proves that: (1) the itaconic acid dimethyl ester can effectively reduce LPS and palmitic acid induced microglial cell inflammation; (2) the dimethyl itaconate can improve cognitive impairment of high-fat diet-induced obese mice; (3) the dimethyl itaconate can improve synaptic ultrastructural damage of high-fat diet-induced obese mice; (4) the dimethyl itaconate can reduce neuroinflammation of high-fat diet-induced obese mice; (5) the dimethyl itaconate has no obvious influence on the weight, the liver, the fat weight, the energy intake and the insulin resistance of the high-fat diet-induced obese mice.

Drawings

FIG. 1 is a schematic representation of the role of itaconic acid in the tricarboxylic acid cycle;

FIG. 2 is a CCK-8 method for detecting the influence of different concentrations of dimethyl itaconate on microglia proliferation and toxicity;^***P<0.001；

FIG. 3 shows that dimethyl itaconate inhibits secretion of TNF- α and IL-6 by LPS-stimulated microglia; A. TNF- α, B, IL-6;^**P<0.01，^***P<0.001；

FIG. 4 shows that dimethyl itaconate inhibits TNF- α and IL-6 secretion from palmitic acid stimulated microglia; A. TNF- α, B, IL-6;^*P<0.05，^***P<0.001；

FIG. 5 is a tear-open condition of cotton in nesting behavioral experiments of mice;

FIG. 6 is a behavioral assessment of nesting in mice; A. counting nesting scores; B. counting the weight of the un-torn cotton;^*P<0.05，^**P<0.01，^***P<0.001；

FIG. 7 is a representation of a mouse object position recognition experiment;

FIG. 8 is a graph of the time to explore new position objects as a percentage of total exploration time in a mouse object position identification experiment;^**P<0.01；

FIG. 9 is a representative diagram of a test for identifying a new mouse;

FIG. 10 is the percentage of time spent exploring new subjects in the mouse new subject trial experiment over the total time explored;^**P<0.01，^***P<0.001；

FIG. 11 is the body weight change of mice; A. body weight curve, B, cumulative body weight change;^***P<0.001；

FIG. 12 shows body weight and liver weight of mice at the time of killing; A. body weight, B and liver when dissected and killedWeighing;^***P<0.001；

FIG. 13 is the change in weight of fat in mice; A. the subcutaneous fat is heavy when killing, the epididymis fat is heavy when killing, and the brown fat is heavy when killing;^*P<0.05，^**P<0.01，^***P<0.001；

FIG. 14 is the energy intake of mice;^**P<0.01；

FIG. 15 is the effect on mouse insulin resistance; A. fasting serum insulin levels; B. HOMA-IR index; C. a glucose tolerance curve; D. area under the glucose tolerance curve;^**P<0.01，^***P<0.001；

FIG. 16 is a diagram of a transmission electron microscope for detecting synapse ultrastructure; in the figure, PSD refers to postsynaptic membrane, AZ refers to active zone, and SC refers to synaptic cleft;

FIG. 17 is a transmission electron microscope used for detecting PSD thickness, active band length and synaptic gap width; A. PSD thickness statistics, B active band length statistics, C synaptic gap width statistics;^*P<0.05，^**P<0.01；

FIG. 18 is a diagram of the detection of SYN protein in the hippocampal region by the Westernblot method; A. an expression representation; B. statistics of SYN protein;^**P<0.01，^***P<0.001；

FIG. 19 is a diagram of the Westernblot method for detecting PSD95 protein in the hippocampus; A. an expression representation; B. statistics of PSD95 protein;^*P<0.05；

FIG. 20 is a diagram of the Westernblot method for detecting hippocampal BDNF protein; A. an expression representation; B. statistics of BDNF proteins;^*P<0.05；

FIG. 21 is an Iba-1 immunofluorescence representation of hippocampal CA1, CA3, DG;

FIG. 22 is Iba-1⁺A map of changes in cells; A. iba-1 in each field of view⁺The number of cells; B. iba-1⁺Circulation analysis of cells; C. iba-1⁺A solid analysis of the cells;^*P<0.05，^**P<0.01，^***P<0.001；

FIG. 23 shows RT-PCR detection of hippocampal CD68 mRNA, TNF-. alpha.mRNA, IL-6mRNA andIL-1 beta mRNA levels; A. CD68 mRNA, B, TNF- α mRNA levels, C, IL-6mRNA levels, D, IL-1 β mRNA levels;^*P<0.05，^**P<0.01，^***P<0.001。

Detailed Description

The present invention is further described below by way of examples, but the present invention is not limited by these examples. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The invention provides an application of dimethyl itaconate in preparing a medicament for treating and/or preventing neurodegenerative diseases. In vitro experiments prove that: the dimethyl itaconate can effectively inhibit microglial cell-mediated neuroinflammation.

The inventor finds in research that dimethyl itaconate can significantly improve obesity-induced cognitive function impairment. In vivo experiments prove that: the dimethyl itaconate can improve the decline of learning, memory and self-care ability of life, improve synaptic ultrastructure, inhibit the activation of microglia and reduce neuroinflammation.

The itaconic acid dimethyl ester can be used for preparing a medicine for preventing, relieving or treating and improving the decline of learning and memory functions.

The dimethyl itaconate can be used for preparing medicines for preventing, relieving or treating and improving synaptic injury.

The itaconic acid dimethyl ester can be used for preparing a medicament for preventing, relieving or treating and improving neuroinflammation.

The itaconic acid dimethyl ester can be used for preparing a medicament for preventing, relieving or treating and improving microglia-mediated neuroinflammation.

The effects of the present invention will be described below with reference to specific experiments.

It should be noted that, the following experimental results are indicated in the corresponding drawings: LC + Vehicle is the normal diet mice injected PBS control group, LC + DI is the normal diet mice injected Dimethyl itaconate (DI, cat # 617-52.7, Sigma-Aldrich) control group, HF + Vehicle is the high fat diet induced obese mice injected PBS group, HF + DI is the high fat diet induced obese mice injected DI group.

First, in vitro experiment

1. Method of producing a composite material

1.1, the microglia line BV-2 is divided into 5X 10⁴And laying the mixture in a 96-well cell culture plate, dividing the plate into a Vehicle control group and a DI group with different concentrations after the cells are attached to the wall, and detecting the proliferation or toxicity of the DI on the BV-2 by using a CCK-8 method after culturing for 24 hours.

1.2, mixing BV-2 at 2X 10⁵After cells were attached to the plates, they were divided into a Vehicle control group, an LPS (100ng/ml) group, a Palmitate (PA, 2.5mM) group, a DI group (0.25. mu.M), a PA + DI group, and an LPS + PA group. After 24h of culture, the secretion of inflammatory factors TNF-alpha and IL-6 in the culture supernatant was examined by ELISA.

2. Results

In the tricarboxylic acid cycle, itaconic acid is produced by cis-aconitate decarboxylase 1 catalyzing the decarboxylation of cis-aconitate (FIG. 1).

2.1 Effect of different concentrations of dimethyl itaconate on the proliferation of BV-2 microglia cells

The CCK-8 method detects the influence and the effect of different concentrations of dimethyl itaconate on the proliferation of BV-2 microglia, and determines that 0.25 mu M is the optimal concentration for stimulating BV-2 cells in vitro (figure 2).

2.2 Effect on LPS and palmitic acid stimulated BV-2 cells

In BV-2 cells stimulated by LPS (bacterial lipopolysaccharide), dimethyl itaconate can remarkably inhibit TNF-alpha and IL-6 secretion of microglia cells (figure 3);

in palmitic acid stimulated BV-2 cells, dimethyl itaconate significantly inhibited the secretion of TNF- α and IL-6 by microglia (FIG. 4).

The above results demonstrate that dimethyl itaconate can significantly inhibit microglial-related neuroinflammation.

Second, in vivo test

1. Mice and groups

Male C57BL/6J mice, 7 weeks old, were purchased from beijing weitongli laboratory animal technology ltd, and after one week of acclimation, were randomly divided into 4 groups of 10 mice each: LC + Vehicle group: routine feed feeding, intraperitoneal injection of 200 μ l sterile PBS solution 2 times per week; LC + DI group: feeding with conventional feed, injecting 2 times per week into abdominal cavity of mice with 200 μ l of PBS solution containing DI (Dimethyl itaconate, 0.5mg/kg, cat # 617-52.7); ③ HF + Vehicle group: feeding with high fat feed, and performing intraperitoneal injection of 200 μ l sterile PBS solution 2 times per week; HF + DI group: mice were fed on a high-fat diet and were intraperitoneally injected 2 times a week with 200. mu.l of a PBS solution containing DI (0.5 mg/kg). The mice were housed in the center of SPF grade animals for 12 weeks. Food intake and weight changes were monitored weekly.

2. Mouse behavioural test

The mice were raised for 12 weeks and then subjected to a study experiment.

2.1 nesting experiment

The mice were placed in cages 1/cage 1h before sundark with padding, one pad (3g) per cage, nesting scores were assessed the next day and unstripped cotton pads (cotton pads greater than 0.1g defined as unstripped) were weighed for assessment of the performance of daily activities of the mice.

Nesting score evaluation criteria:

1 minute: no nesting (more than 90% of cotton remains);

and 2, dividing: forming the nest part (more than 50-90% of cotton is left);

and 3, dividing: the nest is substantially shaped but the nesting location cannot be discerned (50% -90% of the cotton has been shredded, but the cotton is not gathered in the same nest, but is scattered in the cage);

and 4, dividing: nest shaped but flat (more than 90% of the cotton is shredded but the height around the nest is less than 50% of the height of the mouse body);

and 5, dividing: the nest was nearly perfect (more than 90% of the cotton was shredded and the height around the nest was greater than 50% of the height of the mouse body).

2.2 New Object Recognition (NOR) experiment

1 day before the experiment, the mouse is taken to a behavioristics laboratory to be familiar with the environment, and freely explores for 5min in a behavioristics box; after 24h, two identical objects are placed in the behavior box, and exploration is carried out for 5 min; after an interval of 1h, one of the objects was replaced with a new one and was explored for 5 min. The time to explore each object was recorded for evaluation of memory recognition ability of mice.

2.3 object position recognition experiment

1 day before the experiment, the mouse is taken to a behavioristics laboratory to be familiar with the environment, and freely explores for 5min in a behavioristics box; after 24h, putting two identical objects into the behavior box, and exploring for 5 min; after an interval of 1h, one of the objects was moved to a new position and explored for 5 min. The time to explore each object was recorded for evaluation of the spatial memory of the mice.

3. HOMA-IR assay

After fasting for 6 hours, whole blood was collected by a rat tail blood collection method, and fasting blood glucose (mmol/L) and fasting insulin (mU/L) were measured by an ELISA method. A steady-state model insulin resistance index (HOMA-IR), which is the blood glucose concentration (mmol/L) x insulin concentration (mU/L)/22.5, was calculated.

4. Glucose tolerance assay

Before detection, mice are fasted for 16h, and glucose is injected into the abdominal cavity, wherein the injection amount is 1g/kg body weight. Whole blood was obtained by a rat tail blood sampling method, and blood glucose concentrations (mmol/L) were measured with a blood glucose meter at 0, 15min, 30min, 45min, 1h and 2h after injection, respectively.

5. Immunofluorescence

Fresh colon was fixed with 4% by mass of paraformaldehyde for 24 hours, and paraffin-embedded to prepare a paraffin section (5 μm). Sections were dewaxed to water, antigen repaired, blocked with 3% BSA, incubated overnight at ZO-1(ServiceBio, GB11195, 1:200 dilution) and F4/80(ServiceBio, GB11195, 1:1000 dilution) primary antibody at 4 deg.C, incubated with Cy 3-labeled goat anti-rabbit fluorescent secondary antibody at room temperature for 50 minutes in the absence of light, cell nuclei were counterstained with DAPI (ServiceBio, G1012) and incubated for 10 minutes in the absence of light at room temperature. And (4) dropwise adding an autofluorescence quenching agent for 5 minutes, and sealing the anti-fluorescence quenching sealing agent.

After fresh brain tissue is fixed for 24h, sucrose is dehydrated, and after OCT embedding medium is embedded, the fresh brain tissue is sliced by a freezing microtome and has the thickness of 20 mu m. After the water control of the slices, fixing the mass fraction of 4% paraformaldehyde, then performing antigen retrieval, blocking by 3% BSA, and incubating overnight at 4 ℃ for Iba-1(Servicebio, GB13105, 1:50 dilution) and GFAP (Servicebio, GB11096, 1:800 dilution). After washing with PBS, the Cy 3-labeled goat anti-rabbit fluorescent secondary antibody was incubated for 50 minutes in the dark at room temperature, and the cell nuclei were counterstained with DAPI (ServiceBio, G1012) and incubated for 10 minutes in the dark at room temperature. And (4) dropwise adding an autofluorescence quenching agent for 5 minutes, and sealing the anti-fluorescence quenching sealing agent.

The above colon and brain tissue samples were observed using an OLYMPUS IX51 inverted fluorescence microscope and the number of positive cells in the field was counted using ImageJ.

6. Transmission electron microscope

Perfusing the mouse with normal saline and 4% paraformaldehyde by mass fraction, and collecting 1mm of left hippocampi CA1 region³And (4) organizing. After fixation with a mixture of 2% paraformaldehyde and 2.5% glutaraldehyde for 24 hours, PBS was washed three times. With 1% osmium tetroxide (OsO)₄) After 2 hours of postfixation, the sections were then dehydrated in gradient ethanol and acetone, embedded in epoxy resin, and ultrathin sections (70nm) were prepared and stained with 4% uranyl acetate and 0.5% lead citrate. The FEI Tecnai G2SpiritTwin transmission electron microscope is used for measuring the ultrastructure of Gray I type asymmetric synapse, and the shape of the asymmetric synapse is researched. The change of three indexes, namely synaptic activity zone length, postsynaptic compact thickness and synaptic gap width, is analyzed by using Image J software.

7、Westernblot

Cells and tissues were lysed with cell lysates. Total protein concentration was determined using BCA method. The samples were heated in SDS loading buffer at 95 ℃. The fractions were fractionated using a 10% SDS-PAGE gel and then transferred to a PVDF membrane. BDNF, PSD95, SYN and β -Actin antibodies were used overnight at 4 ℃ in TBST with 1% skim milk. The secondary antibody was incubated for 1 h. And preparing and uniformly mixing the chemiluminescence solution A and the chemiluminescence solution B according to the volume of 1: 1. The chemiluminescent liquid is completely covered on the membrane, and the gel electrophoresis imaging analyzer is used for photographing and analyzing.

8. Fluorescent quantitative RT-PCR

Hippocampus RNA was extracted by Trizol method, reverse transcribed, and detected using Roche LightCycler 480II fluorescent quantitative PCR instrument. Using equation 2^-ΔΔCtCalculating the mRNA level of a specific gene and using beta-actiLevels of nmRNA were normalized. The fluorescence signal is generated by a fluorescent DNA binding dye (SYBR Green I). Fluorescence quantitative PCR machine program: pre-denaturation: 95 ℃ for 5 minutes. Amplification cycle: denaturation: 95 ℃ for 15 seconds. Annealing: 60 ℃ for 15 seconds. Extension: 72 ℃ for 15 seconds. For a total of 45 cycles. Melting procedure: 95 ℃ for 5 seconds, 65 ℃ for 1 minute, and then heated to 97 ℃ to denature the DNA product. Cooling program: 30 seconds at 40 ℃. The primer sequences are shown in Table 1.

TABLE 1 fluorescent quantitative PCR primer sequences

Name of the lead	Upstream sequence (Forward)	Downstream sequence (Reverse)
			TNF-α	CTTGTTGCCTCCTCTTTTGCTTA	CTTTATTTCTCTCAATGACCCGTAG
IL-6	TCACAGAAGGAGTGGCTAAGGACC	ACGCACTAGGTTTGCCGAGTAGAT
			IL-1β	TGGGAAACAACAGTGGTCAGG	CTGCTCATTCACGAAAAGGGA
CD68	TCACCTTGACCTGCTCTCTCTAA	GCTGGTAGGTTGATTGTCGTCTG
			β-actin	CGTGGGCCGCCCTAGGCACCA	TTGGCCTTAGGGTTCAGGGGGG

9. Statistical analysis

Normally distributed metric data andand (4) showing. Through the test of normality and homogeneity of variance, two groups of comparison with normality and homogeneity of variance adopt t test, the difference between a plurality of groups of measurement data adopts one-factor analysis of variance (ANOVA), pairwise comparison among all groups adopts Turkey's method test, wherein a plurality of experimental groups and a control group are compared and adopt Dunnett method test. The test level α is 0.05. Statistical analysis was performed using SPSS 21.0 software.

10. Results

10.1, dimethyl itaconate can improve cognitive impairment induced by high fat diet in obese mice

In the nesting experiment, dimethyl itaconate can significantly improve the nesting score of obese mice and reduce the weight of undrawn cotton (fig. 5-6), indicating that dimethyl itaconate can improve the self-care ability of the obese mice.

In the subject position trial experiments, the percentage of time that the dimethyl itaconate intervened in the search of obese mice for new position subjects was significantly increased compared to the solution control group for the total search time (fig. 7-8).

In the neosome experiments, the percentage of the time during which dimethyl itaconate intervenes in the search for neosome in obese mice was significantly increased compared to the total search time (fig. 9-10).

The research results show that the dimethyl itaconate can improve the self-care, learning and memory abilities of high-fat diet-induced obese mice.

10.2 Effect of dimethyl itaconate on high fat diet induced weight, liver, fat weight, energy intake, insulin resistance in obese mice

Dimethyl itaconate did not improve body weight and weight gain in obese mice (figure 11). At the time of killing, the weight, liver weight, subcutaneous fat, epididymal fat and brown fat of the obese mice were not significantly changed compared to the control obese mice (fig. 11-13), and daily energy intake was not significantly changed (fig. 14).

Dimethyl itaconate did not significantly improve fasting serum insulin levels and HOMA-IR index in obese mice relative to control obese mice (fig. 15A-B); but can significantly affect the glucose tolerance profile in obese mice (fig. 15C-D).

10.3, the itaconic acid dimethyl ester can improve synaptic ultrastructural damage of fat mice induced by high fat diet

The transmission electron microscope detection result shows that the dimethyl itaconate can obviously increase the thickness of postsynaptic membrane protein PSD95, increase the length of an active band and reduce the width of a synaptic cleft in an obese mouse (fig. 16-17).

Westernblot showed that compared with control obese mice, dimethyl itaconate intervened in the expression of presynaptic membrane protein (SYN), postsynaptic membrane protein PSD95 and neurotrophic factor BDNF in hippocampal region of obese mice to be obviously up-regulated (FIGS. 18-20).

These results indicate that dimethyl itaconate is effective in preventing high fat diet-induced synaptic ultrastructural damage.

10.4 dimethyl itaconate can reduce neuroinflammation of high fat diet induced obese mice

Compared with obese control mice, the dimethyl itaconate intervenes in Iba-1 in hippocampal CA1, CA3 and DG of the mice⁺The number of cells (microglia) was significantly reduced (fig. 21), and the circulation, solubility index of the cells was significantly reduced (fig. 22). The dimethyl itaconate can inhibit the increase and activation of the number of microglia.

Meanwhile, dimethyl itaconate intervenes in the significant down-regulation of mRNA levels of CD68, TNF-alpha, IL-6 and IL-IL-1 beta in hippocampal region of obese mice (FIG. 23).

These results indicate that dimethyl itaconate can inhibit the activation of microglia in obese mice, thereby ameliorating neuroinflammation.

In conclusion, dimethyl itaconate can improve obesity-induced cognitive impairment, synaptic ultrastructural damage and neuroinflammation.

The above disclosure is only for the specific embodiment of the present invention, but the embodiment of the present invention is not limited thereto, and any variations that can be made by those skilled in the art should fall within the scope of the present invention.