Application of ethanol to reduction of amyloid protein deposition

Abstract

The invention relates to the technical field of biological medicines, in particular to application of ethanol to reduction of amyloid protein deposition. The application comprises application of ethanol in reduction of amyloid protein deposition; the invention relates to an application of ethanol in inhibition of APOE3 allele expression. The invention relates to an application of ethanol in reducing APOE3 protein. According to the present invention, the APOE3 allele is inhibited by using ethanol to express the APOE3 protein so as to reduce the amyloid protein deposition, such that the new lifestyle is provided for the AD high risk population so as to effectively delay the pathological process, create the idea for the new diagnosis and treatment means, and lay the foundation for the AD clinical treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of ethanol in reducing amyloid protein deposition. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease among age-dependent dementias. ApoE is the main susceptibility gene for late-onset AD. ApoE is a glycoprotein mainly produced by the liver and is a lipid transport protein. The brain is the second largest source of apoE. [GUO T,ZHANG D,ZENG Y,et al.Molecular and cellular mechanisms underlying the pathogenesis of Alzheimer's disease[ J].Mol Neurodegener,2020,15(1):40.] In the brain, APOE is highly expressed in astrocytes, microglia, and neurons under stress [HU T,WANG S,CHEN C,et al.Real-Time Analysis of Binding Events between Different Aβ(1-42)Species and Human Lilrb2 by Dual Polarization Interferometry[J].Anal Chem,2017,89(4):2606-12] , responsible for transporting cholesterol and phospholipids synthesized by glial cells to neurons. APOE has three subtypes: APOE2 (ε2), APOE3 (ε3) and APOE4 (ε4). Among them, APOE3 is the most common phenotype in the population.

[0003] However, there are different views on the relationship between drinking and human health. Ethanol molecules are non-polar and can pass through the blood-brain barrier (BBB) ​​without any obstacles. The brain is one of the important target organs of alcohol, so the effect of alcohol on the nervous system is particularly important. Many studies have shown that long-term or heavy drinking can lead to central and peripheral nervous system diseases. [SCHUCKIT M A.Alcohol,Anxiety,and Depressive Disorders[J].Alcohol Health Res World,1996,20(2):81-5] However, in China, it is popular to say that "moderate drinking is good for health", and many studies have shown that moderate drinking is good for human health. [2016Alzheimer's disease facts and figures[J].Alzheimers Dement,2016,12(4):459-509.] , including the prevention and control of depression, cardiovascular and cerebrovascular diseases, etc. Studies have shown that low doses of ethanol can enhance the function of the glymphatic system [LUNDGAARD I,WANG W,EBERHARDT A,et al.Beneficial effects of low alcohol exposure,but adverse effects of high alco hol intake on glymphatic function[J].Sci Rep,2018,8(1):2246] , thereby alleviating AD pathology.

[0004] The present invention studies the relationship between ethanol intake and the APOE3 allele, and finds that in a mouse experiment, alcohol feeding during adolescence and adulthood can effectively reduce the deposition of 5×FAD / APOE3+ / + amyloid plaques. Therefore, the application of ethanol in reducing amyloid protein deposition is proposed, which provides a new idea for AD pathology research. Summary of the invention

[0005] The purpose of the present invention is to provide an application of ethanol in reducing amyloid protein deposition, so as to develop a new method for reducing amyloid protein deposition by inhibiting the expression of APOE3 protein by APOE3 alleles, thereby proposing a new idea for AD pathology research.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides application of ethanol in reducing amyloid protein deposition.

[0008] The present invention provides application of ethanol in inhibiting APOE3 allele from expressing APOE3 protein.

[0009] The present invention has at least the following beneficial effects:

[0010] The present invention provides a method of inhibiting the expression of APOE3 protein by APOE3 allele through ethanol, thereby reducing amyloid protein deposition, providing a new lifestyle for people at high risk of AD, thereby effectively delaying the pathological process and creating ideas for new diagnosis and treatment methods, and also laying a foundation for the clinical treatment of AD. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0012] Figure 1 Schematic diagram of the comparison of amyloid plaques in the 5×FAD / APOE3+ / + treatment groups and the control group by immunofluorescence;

[0013] Figure 2 This is a schematic diagram comparing the deposition of β-amyloid protein in the 5×FAD / APOE3+ / + treatment groups and the control group by immunofluorescence. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0015] The main purpose of the present invention is to provide an application of ethanol in reducing amyloid deposition, which is specifically as follows:

[0016] 1. Choose an appropriate alcohol feeding model.

[0017] In previous studies, there are many models commonly used to study alcoholic liver injury in mice. After balancing the research purpose and the existing laboratory equipment, a chronic ethanol feeding model with ethanol added to drinking water as the only water source for a long time was selected. This method will cause many immune abnormalities and mild fatty degeneration, but has little effect on serum alanine aminotransferase, aspartate aminotransferase and liver inflammation. Therefore, this model can be used to explore neurological diseases and effectively exclude the impact of other diseases caused by drinking on mouse pathology.

[0018] 2. Establishment of AD mouse models and control models of chronic alcohol drinking at different growth stages

[0019] The laboratory introduced the mixed mouse model of Aβ plaques and CAA 5×FAD / APOE4 from David Holtzman's laboratory + / + As a control, 5×FAD mice were crossed with APOE3 mice to obtain 5×FAD / APOE3 + / + Double gene transgenic mice simulate the most common subtype of APOE in the human population, APOE3. + / + The experimental mice were divided into three groups: embryonic, adolescent and adult. The three groups of mice were fed with 5% chronic ethanol for 21 days from embryonic day 1 to day 21 (ethanol treatment for parents), 0.75 months of age (adolescence) and 2 months of age (adulthood), and the control group was given normal water as a control. All animals were housed individually in standard mouse cages with an artificial light-dark cycle of 12 hours, and the room temperature and humidity were kept constant. Standard laboratory mouse chow and drinking water or 5% ethanol were provided unlimited during the experiment. The brain was removed at 4.5 months of age for pathophysiological analysis.

[0020] 3. Mouse brain tissue extraction

[0021] (1) Infusion

[0022] 1) Anesthesia: 5% chloral hydrate was injected into the mouse at a ratio of 100 ul / g by intraperitoneal injection. After the mouse's respiratory rate stabilized, the limbs were fixed, the chest cavity was cut open to expose the heart, and a needle that had been rinsed with 0.5 M EDTA was pointed at the apex of the heart to draw apical blood. The blood was centrifuged at 5,500 g for 10 minutes at 4°C, and the supernatant was obtained as a plasma sample, which was stored at -80°C for later use.

[0023] 2) Use a peristaltic pump to perfuse pre-chilled phosphate buffered saline (PBS) at a flow rate of 11 ml / min for 3 minutes until the liver turns white, then stop perfusion.

[0024] 3) Anesthetize the mice by intraperitoneal injection of 5% chloral hydrate at a ratio of 100 ul / g. After the respiratory rate of the mice stabilized, the limbs were fixed, the chest cavity was cut open to expose the heart, and the apical blood was drawn by aligning a needle that had been rinsed with 0.5 M EDTA in advance to the apex. The blood was centrifuged at 5,500 g for 10 minutes at 4°C. The supernatant was obtained as a plasma sample and stored at -80°C for later use.

[0025] 4) Use a peristaltic pump to perfuse pre-chilled phosphate buffered saline (PBS) at a flow rate of 11 ml / min for 3 minutes until the liver turns white, then stop the perfusion.

[0026] (2) Tissue fixation and dehydration

[0027] 1) Carefully remove the mouse brain with forceps and place it on an ice plate. The left half of the brain is placed in pre-cooled 4% paraformaldehyde (PFA) at 4°C overnight. The other half is peeled off according to the location of different brain regions, such as the anterior and posterior cortex, hippocampus, cerebellum, thalamus, etc., and immediately frozen at -80°C for later use.

[0028] 2) The left brain was treated with 4% PFA at 4°C overnight and then transferred to 25% sucrose overnight. The next day, the brain was transferred to 30% sucrose until it was completely precipitated. The slices were cut using a Leica slicer and placed in a pre-prepared freezing solution at -20°C for later use.

[0029] The effects of alcohol feeding on the pathological changes of AD mice will be described in detail below with reference to the accompanying drawings.

[0030] See also Figure 1 , Figure 1 The amyloid plaques in the 5×FAD / APOE3+ / + treatment group and the control group were compared by immunofluorescence. The plaques formed by amyloid protein deposition are one of the most important pathological manifestations of AD. Figure 1Middle: (A) X-34 (white) staining of 5×FAD / APOE3+ / + female mouse control group, gestational alcohol feeding group, adolescence alcohol feeding group, and adulthood alcohol feeding group, 20×, scale bar = 500μm; (B) The percentage of amyloid plaque area in the cortex of 5×FAD / APOE3+ / + adolescent alcohol feeding group and adulthood alcohol feeding group decreased compared with the drinking water control group, n = 5-8 for each group; (C) X-34 (white) staining of 5×FAD / APOE3+ / + male mouse control group, gestational alcohol feeding group, adolescence alcohol feeding group, and adulthood alcohol feeding group, 20×, scale bar = 500μm; (D) The percentage of amyloid plaque area in the cortex of 5×FAD / APOE3+ / + gestational alcohol feeding group, adolescence alcohol feeding group, and adulthood alcohol feeding group decreased compared with the drinking water control group, but no significant difference was observed, n = 5-8 for each group. A brain slice from a different position was selected for each mouse, and the same position was selected for each group. The final result was averaged and Student's t test was used. The experimental data are expressed as ±SEM, *P<0.05, **P<0.01, ***P<0.001.

[0031] We selected two brain slices of the same unprecedented dimension for each mouse for X-34 staining to represent the deposition of amyloid plaques. We distinguished the mice by gender and made fine regional divisions. The percentage of X-34 positive signal coverage in the cortex, the most typical area for amyloid plaque deposition, was statistically analyzed. Our results showed that in female 5×FAD / APOE3+ / + mice, compared with the control group of water-fed mice, the amyloid plaques in the cortex of mice fed with alcohol during adolescence and alcohol during adulthood were greatly reduced, especially in the adult alcohol-fed group. Male 5×FAD / APOE3+ / + mice also had a downward trend, but no significant difference was seen. However, no significant difference was seen in 5×FAD / APOE4+ / + mice. This shows that alcohol feeding during adolescence and adulthood can effectively reduce the deposition of 5×FAD / APOE3+ / + amyloid plaques, and this downward trend is correlated with APOE subtype and gender.

[0032] See also Figure 2 , Figure 2 The deposition of β-amyloid protein in the 5×FAD / APOE3+ / + treatment group and the control group was compared by immunofluorescence. Figure 2Middle: (A) HJ3.4 (red) staining of 5×FAD / APOE3+ / + female mouse control group, gestational alcohol-fed group, adolescence alcohol-fed group, and adulthood alcohol-fed group, 20×, scale bar = 500 μm; (B) The area percentage of β-amyloid protein in the cortex of 5×FAD / APOE3+ / + adolescent alcohol-fed group and adulthood alcohol-fed group decreased compared with the drinking water control group, n = 5-8 for each group; (C) HJ3.4 (red) staining of 5×FAD / APOE3+ / + male mouse control group, gestational alcohol-fed group, adolescence alcohol-fed group, and adulthood alcohol-fed group, 20×, scale bar = 500 μm; (D) The area percentage of β-amyloid protein in the cortex of 5×FAD / APOE3+ / + gestational alcohol-fed group, adolescence alcohol-fed group, and adulthood alcohol-fed group decreased compared with the drinking water control group, but no significant difference was observed, n = 5-8 for each group. A brain slice from a different position was selected for each mouse, and the same position was selected for each group. The final result was averaged and Student's t test was used. The experimental data are expressed as ±SEM, *P<0.05, **P<0.01, ***P<0.001.

[0033] Here, we purified the HJ3.4 hybridoma cell line from David Holtzman's laboratory at the University of Washington School of Medicine and used the HJ3.4 antibody to identify Aβ in the brain. We found that the Aβ coverage area in the brain of each group of mice was Figure 1 The deposition trend of amyloid plaques echoed that of the control group, verifying the credibility of the results.

[0034] (1) Immunofluorescence

[0035] 1) Use a flat-push cryostat to slice each piece into 50 μm thick slices, place them in cryopreservation solution, and store them at -20°C.

[0036] 2) Pick out the required brain slices and place them in a twelve-well plate containing PBS to rewarm for 10 minutes, then wash them with PBS three times, 5 minutes each time.

[0037] 3) Add 2 ml of pre-prepared PBS permeabilization solution (PBS + 0.25% Triton X-100) to each well and incubate on a shaker at room temperature for 20 minutes.

[0038] 4) Wash three times with PBS for five minutes each time.

[0039] 5) Perform X-34 staining and incubate the prepared X-34 staining solution (40% ETOH, 60% PBS, 1:500 10NNaOH, 1:5,000 X-34) at room temperature for 20 minutes.

[0040] 6) Wash three times with 40% ETOH + 60% PBS, each time for 2 minutes.

[0041] 7) Wash with PBS three times for 5 minutes each time.

[0042] 8) Add 2 ml of blocking solution (5% BSA+PBST) to each well and block for 1 hour at room temperature.

[0043] 9) Dilute the corresponding primary antibody with blocking solution and incubate the brain slices in a 48-well plate at 4°C overnight.

[0044] 10) The next day, pick out the brain slices that have been incubated overnight and wash them three times with PBS for 5 minutes each time.

[0045] 11) The following steps were kept away from light. The corresponding secondary antibody was used to incubate in a 48-well plate at room temperature for 1 hour, then washed three times. The brain slices were placed on a glass slide in PBS and air-dried.

[0046] 12) After the brain slices are completely air-dried, wash them three times in dd water and then air-dry them naturally.

[0047] 13) Using Thermo Fisher's ProLong TM Gold was used for sealing.

[0048] (2) Laser disk confocal imaging

[0049] All fluorescent staining in this section was photographed by Olympus' laser spinning disk confocal. The shooting magnification was 20 times, the depth was 20um, a total of 10 layers, and Z stacking.

[0050] All fluorescent slides were scanned within one week using a VS200 whole slide scanner or laser spinning disk confocal.

[0051] Before analyzing the data, a normality test was performed. Statistical analysis was performed using Student's t test and one-way analysis of variance (ANOVA). If the data did not meet the requirement for homogeneity of variance, the Welch correction was used for testing. All statistics were two-tailed tests, and the experimental data were expressed as mean ± standard error (mean ± SEM). Immunofluorescence statistics were performed using Image J software, and statistical analysis and graph drawing were performed using GraphPad Prism 9. *P < 0.05 was considered statistically significant.

[0052] Therefore, combined with the above content, it can be seen that:

[0053] The present invention discloses inhibiting the APOE3 allele from expressing the APOE3 protein, thereby reducing the deposition of amyloid protein.

[0054] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions only describe the principles of the present invention. The present invention may be subject to various changes and improvements without departing from the spirit and scope of the present invention. These changes and improvements fall within the scope of the present invention. The scope of protection claimed by the present invention is defined by the attached claims and their equivalents.

Claims

1. Application of ethanol in reducing amyloid protein deposition.

2. Application of ethanol in inhibiting the expression of APOE3 protein by APOE3 allele.

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