Application of folic acid in preparation of medicine for improving related learning and memory impairment caused by synaptic plasticity abnormality

Abstract

The invention relates to the field of pharmacy, in particular to application of folic acid in preparation of a medicine for improving related learning and memory impairment caused by synaptic plasticity abnormity. By exploring the molecular mechanism of the hippocampal synaptic plasticity injury caused by fluorine-aluminum combined exposure, it is found that reduction of the methylation level of the genome is one of the mechanisms of the hippocampal synaptic plasticity injury caused by fluorine-aluminum combined exposure. After folic acid is added, the expression level of synaptic proteins SYP and PSD-95, the methylation level of genomic DNA and the expression level of related methylated transferases DNMTI, DNMT3a and DNMT3b can be improved, and it is indicated that folic acid can improve synaptic plasticity damage caused by fluorine-aluminum combination by improving the methylation level of the genomic DNA; the compound can be used for preparing medicines for treating or improving related learning and memory impairment caused by synaptic plasticity abnormity caused by fluorine-aluminum combination.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to the application of folic acid in the preparation of a drug for improving learning and memory impairment caused by abnormal synaptic plasticity. Background Art

[0002] Fluoride is extremely active in chemical properties and exists mainly in the form of compounds in the environment. It can enter the human body through drinking water, tea, food, and environmental media in industrial pollution areas. It is beneficial to the human body in moderate amounts, but excessive amounts can lead to fluoride poisoning. Currently, fluoride has been listed as a developmental neurotoxin. Aluminum is a recognized neurotoxin that is associated with the occurrence and development of neurodegenerative diseases such as Alzheimer's disease accompanied by cognitive impairment.

[0003] At present, most of the research reports on the toxicity of fluorine and aluminum are mainly based on single poisoning, but the health effects of co-exposure to chemicals in the environment cannot be ignored. Excessive fluorine and aluminum coexist in industrial waste gas pollution areas where aluminum is produced by cryolite-alumina molten salt electrolysis, coal-burning type fluorosis areas where kaolin is mixed with coal, brick tea type fluorosis areas, and drinking water. Fluorine and aluminum can form fluorine-aluminum complexes in the gastrointestinal tract. Fluoride increases the ability of aluminum to pass through the blood-brain barrier, enhances neuronal oxidative stress, causes lipid peroxidation damage, changes synaptic structure and function, and reduces synaptic transmission efficiency; it can also lead to a significant decrease in neuronal density and damage neurons; fluorine, aluminum and their complexes can penetrate the placenta, easily reach the fetal circulation, cross the underdeveloped blood-brain barrier, interfere with the normal function of brain neurons, and cause brain development disorders, manifested as cognitive dysfunction.

[0004] Synaptic plasticity is one of the hot topics in cognitive dysfunction research in recent years. It is closely related to learning and memory ability. Under pathological conditions, it manifests as abnormal synaptic plasticity of hippocampal neurons, including degeneration and loss of neuronal synapses, which impairs the learning and memory process. Abnormal synaptic plasticity is an important mechanism for learning and memory disorders caused by co-exposure to fluorine and aluminum and many neurological diseases. There is currently a lack of therapeutic drugs and methods developed for such diseases. Therefore, it is necessary to develop drugs for treating or improving learning and memory impairments associated with abnormal synaptic plasticity. Summary of the invention

[0005] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide the use of folic acid in the preparation of drugs for improving learning and memory impairment caused by abnormal synaptic plasticity. The present invention verifies through experiments that folic acid can significantly increase the expression levels of synaptic proteins SYP and PSD-95, the genomic DNA methylation level and the expression levels of related methyltransferases DNMTI, DNMT3a, and DNMT3b; folic acid can improve the synaptic plasticity damage caused by the combination of fluorine and aluminum by increasing the genomic DNA methylation level, and can be used to prepare drugs for improving learning and memory impairment caused by abnormal synaptic plasticity.

[0006] The present invention provides an application of folic acid in the preparation of a drug for improving learning and memory impairment caused by abnormal synaptic plasticity.

[0007] Furthermore, the folic acid is used to prepare a drug for improving learning and memory impairment caused by abnormal synaptic plasticity due to combined exposure to fluorine and aluminum.

[0008] Furthermore, the drug contains folic acid as the only active ingredient.

[0009] Furthermore, the drug also contains pharmaceutically acceptable excipients and / or carriers.

[0010] Furthermore, the dosage form of the drug includes tablets, powders, granules or injections.

[0011] Furthermore, the auxiliary material is any one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, a matrix and an antioxidant.

[0012] Furthermore, the injection is a solution or an emulsion.

[0013] Furthermore, the folic acid is used to prepare an expression promoter of synaptic protein SYP or PSD-95.

[0014] Furthermore, the promoter for increasing the expression of synaptic protein SYP or PSD-95 is used to prepare a drug for promoting neural remodeling and repair.

[0015] In summary, compared with the prior art, the present invention has the following advantages and effects: The present invention discloses for the first time the use of folic acid in the preparation of a drug for improving learning and memory impairment caused by abnormal synaptic plasticity. The folic acid provided by the present invention can significantly increase the expression levels of synaptic proteins SYP and PSD-95, the genomic DNA methylation level and the expression levels of related methyltransferases DNMTI, DNMT3a, and DNMT3b; neuronal synaptic plasticity is the biological basis of learning and memory. In many nervous system diseases, the combination of fluorine and aluminum can cause synchronous impairment of synaptic function and memory. The folic acid provided by the present invention can improve the treatment of learning and memory impairment by repairing synaptic communication. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The effect of combined exposure to fluorine and aluminum on the expression of SYP and PSD-95 mRNA in the hippocampus of the second-generation mice; a indicates a comparison with the control group, p <0.05.

[0017] Figure 2 The effect of combined exposure to fluoride and aluminum on the expression of SYP and PSD-95 proteins in the hippocampus of the second-generation offspring mice; Figure 2 Figure A and Figure 2 Figure B shows the effects on the expression levels of SYP and PSD-95 proteins. Figure 2 Figure C is the protein gel electrophoresis of SYP and PSD-95; a indicates that compared with the control group, p <0.05.

[0018] Figure 3 The effect of combined exposure to fluorine and aluminum on the whole genome DNA methylation level in the hippocampus of the second-generation mice; a indicates that compared with the control group, p <0.05.

[0019] Figure 4 The effect of combined exposure to fluorine and aluminum on the mRNA expression of DNMT1, DNMT3a and DNMT3b in the hippocampus of the second-generation mice; a indicates a comparison with the control group, p <0.05.

[0020] Figure 5 To investigate the effects of combined fluoride and aluminum exposure on the expression of DNMT1, DNMT3a and DNMT3b proteins in the hippocampus of second-generation offspring mice; Figure 5 Figure A Figure 5 Figure B and Figure 5 Figure C shows the effects on the protein expression levels of DNMT1, DNMT3a, and DNMT3b; Figure 5 Figure D is the protein gel electrophoresis diagram of DNMT1, DNMT3a and DNMT3b; a indicates that compared with the control group, p <0.05.

[0021] Figure 6 This is the effect of folic acid on the viability of NG108-15 cells.

[0022] Figure 7 is the effect of folic acid on the viability of NG108-15 cells exposed to fluorine and aluminum. a means compared with the control group. p <0.05; b means compared with the corresponding general exposure group, p <0.05.

[0023] Figure 8 The effect of folic acid on the expression of SYP and PSD-95 mRNA in NG108-15 cells exposed to fluorine and aluminum; a means compared with the control group, p <0.05; b means compared with the corresponding general exposure group, p <0.05.

[0024] Fig. 9 The effect of folic acid on the expression of SYP and PSD-95 proteins in NG108-15 cells exposed to fluoride and aluminum; Fig. 9 Figure A and Fig. 9 Figure B shows the effects on the expression levels of SYP and PSD-95 proteins. Fig. 9 Figure C is the protein gel electrophoresis of SYP and PSD-95; a indicates that compared with the control group, p <0.05; b means compared with the corresponding general exposure group, p <0.05.

[0025] Fig.10 is the effect of folic acid on the whole genome DNA methylation level of NG108-15 cells exposed to fluorine and aluminum; a means compared with the control group, p <0.05; b means compared with the corresponding general exposure group, p <0.05.

[0026] Fig.11 The effect of folic acid on the mRNA expression of DNMT1, DNMT3a and DNMT3b in NG108-15 cells exposed to fluorine and aluminum. a indicates that compared with the control group, p <0.05; b means compared with the corresponding general exposure group, p <0.05.

[0027] Fig.12 The effect of folic acid on the expression of DNMT1, DNMT3a and DNMT3b proteins in NG108-15 cells exposed to fluoride and aluminum; Fig.12 Figure A Fig.12 Figure B and Fig.12 Figure C shows the effects on the protein expression levels of DNMT1, DNMT3a, and DNMT3b; Fig.12 Figure D is the protein gel electrophoresis diagram of DNMT1, DNMT3a and DNMT3b; a means compared with the control group, p <0.05; b means compared with the corresponding general exposure group, p <0.05. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0029] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0030] With the continuous deepening of research on endemic fluorosis and trace elements, the study of the neurotoxic effects of fluoride and aluminum has attracted more and more attention. Synaptic plasticity is one of the hot topics in the study of cognitive dysfunction in recent years. It is closely related to learning and memory ability. Under pathological conditions, it manifests as abnormal synaptic plasticity of hippocampal neurons, including degeneration and loss of neuronal synapses, which impairs the learning and memory process. Abnormal synaptic plasticity is an important mechanism for learning and memory disorders caused by co-exposure to fluoride and aluminum and many neurological diseases. There is currently a lack of therapeutic drugs and means developed for such diseases. Therefore, it is necessary to develop drugs for the prevention and treatment of learning and memory damage related to abnormal synaptic plasticity.

[0031] The present invention provides an application of folic acid in the preparation of a drug for improving learning and memory impairment caused by abnormal synaptic plasticity. The present invention simulates the actual situation of continuous exposure of children living in a fluorine-aluminum coexistence environment, establishes a second-generation offspring mouse animal model of fluorine-aluminum combined exposure, explores the mechanism of synaptic plasticity damage of hippocampal neurons caused by fluorine-aluminum combined exposure, and then establishes a fluorine-aluminum combined poisoning cell model with or without folic acid intervention, and detects the effect of folic acid on improving learning and memory impairment caused by abnormal synaptic plasticity.

[0032] Example 1: Mechanism of synaptic plasticity damage caused by combined exposure to fluorine and aluminum 1. Experimental Methods 1. Establishment of a second-generation mouse model of continuous fluoride and aluminum combined exposure (1) Subjects and feeding conditions Sixteen healthy clean grade SD pregnant mice, weighing 220-250 g, were provided by the Animal Experiment Center of Guizhou Medical University. The facility certificate number is SYXK (Guizhou) 2018-0001, and the animal quality certificate number is SCXK (Guizhou) 2018-0001. This experiment was approved by the Ethics Committee of Guizhou Medical University (No. 1603184). During the experiment, the rats had free access to water and food. The feeding conditions were as follows: room temperature of 20 °C, relative humidity of 60%-70%, and light of 278 lx.

[0033] (2) Basis for dose selection, animal grouping and treatment ①Acute toxicity LD of sodium fluoride (NaF) 50 is 147.5 mg / kg (rat oral). According to the toxicological design principles and the previous research results of the inventor's research group, the toxic dose of NaF was determined to be (1 / 10 LD 50 ) 120 mg / L; According to the weekly allowable intake of aluminum in food of 7.0 mg / kg and the safety factor between humans and animals, aluminum trichloride (AlCl 3 ) is 600 mg / L (1 / 10 LD 50 The pregnant mice were randomly divided into 4 dose groups according to their body weight: control, single fluoride, single aluminum and fluoride and aluminum combined groups. The drinking water of each group contained NaF and AlCl 3 The final concentrations were 0mg / L+0mg / L, 120mg / L+0mg / L, single aluminum group 0mg / L+600mg / L and 120mg / L+600mg / L respectively.

[0034] ② Free drinking water exposure, parental rats were exposed to poison from pregnancy to postnatal day 21 (PND21) of the first generation of offspring. After weaning and separation, 8 first generation offspring were randomly selected from each group according to body weight (female: male ratio was 1:1) and continued to be treated with the same dose and method until sexual maturity (90 days). 4 female rats and 2 male rats (female: male ratio was 2:1) were randomly selected from each group according to body weight and mated in the same cage. The treatment method was the same as before. The first generation of offspring was exposed from pregnancy day 0 to the 21st day of the second generation of offspring; 8 second generation offspring were randomly selected from each group according to body weight (female: male ratio was 1:1) and continued to be exposed to the same dose and method until the 8th week after birth. Weigh once every 2 weeks.

[0035] ③After the poisoning, the animals were killed by isoflurane anesthesia, and the hippocampus was separated from the brain tissue, divided into cryopreservation tubes, and stored in a -80℃ refrigerator for future use.

[0036] 2. ELISA detection of whole genome DNA methylation level in hippocampal tissue (1) Extraction of genomic DNA from hippocampal tissue Genomic DNA from hippocampal tissue was extracted according to the instructions of the genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).

[0037] (2) Detection of whole genome DNA methylation levels The whole genome DNA methylation level of hippocampal tissue was detected according to the instructions of the 5-mC DNA ELISA kit (purchased from ZYMO RESEARCH, USA), and the content of 5-mC was determined using an enzyme reader.

[0038] 3. Real-time fluorescence quantitative (qRT-PCR) detection of mRNA expression levels of SYP, PSD-95, DNMT1, DNMT3a and DNMT3b in hippocampal tissue (1) Total RNA extraction RNA from hippocampal tissue was extracted according to the instructions of Trizol Reagent RNA extraction reagent (purchased from Invitrogen, USA) and stored in a -80°C refrigerator for later use. The RNA concentration was determined using an ELISA reader.

[0039] (2) Reverse transcription of total RNA into cDNA Add 1 μL dNTP, 0.5 μL oligo(dT), and 1 μg Total RNA to an EP tube, add DEPC water to make up to 10 μL, centrifuge for 30 seconds, and then perform reverse transcription. Set the reverse transcription program on the PCR instrument as follows: 65 °C for 5 min, 4 °C for 2 min, and obtain system I. After adding 10.0 μL system I, 0.5 μL RRI, 0.5 μL M-MLV, 4 μL 5× buffer, and 5.0 μL DEPC water into a 100 μL EP tube, centrifuge for 30 s for reverse transcription. The reverse transcription program of the PCR instrument was set as follows: 42 ℃ 30 min, 75 ℃ 15 min, and 4 ℃ 2 min to obtain cDNA.

[0040] (3) qRT-PCR detection of SYP, PSD-95, DNMT1, DNMT3a and DNMT3b mRNA expression levels ① Primer synthesis: SYP, PSD-95, DNMT1, DNMT3a, DNMT3b and GAPDH (internal reference) primers were designed and synthesized by Shanghai Jierui Biotechnology Co., Ltd. The specific information is shown in Table 1.

[0041] Table 1 qRT-PCR primer sequences ②The qRT-PCR reaction system is shown in Table 2: Table 2 Reaction system ③ The reaction procedure was: 95℃ 30s; 95℃ 5s, 60℃ 30s, 72℃ 30s, after 40 cycles, 2 -ΔΔCt The relative expression of mRNA was calculated by the method.

[0042] 4. Western Blotting (WB) to detect the protein expression levels of SYP, PSD-95, DNMT1, DNMT3a and DNMT3b in hippocampal tissue Take the hippocampal tissue, weigh it, and put it in a grinding tube containing grinding beads; use a pipette to add 140μL of pre-cooled (4℃) protein lysis buffer; grind the tissue into tissue homogenate with a grinder, and transfer the homogenate into an EP tube with a pipette (the above procedures are all performed on ice), and centrifuge it at 12000 r / min for 15 min at 4℃; take the supernatant after centrifugation, quantify the prepared protein using the BCA method, add PBS and 5×SDS loading buffer to adjust the sample to the same protein concentration, boil it in a boiling water bath for 10 min, and then store it at -80℃ for later use.

[0043] Protein electrophoresis was performed on SYP, PSD-95, DNMT1, DNMT3a and DNMT3b. After development with developer, the optical density values ​​were calculated using Image-J software and statistical analysis was performed.

[0044] 2. Experimental Results 1. Determination of the effects of combined fluoride and aluminum exposure on the expression levels of SYP and PSD-95 SYP and PSD-95 are specific marker proteins of synaptic plasticity. SYP is located at the presynaptic end and synaptic vesicles. It is one of the main components of the presynaptic membrane and belongs to the synaptic vesicle membrane protein that transports calcium ions and neurotransmitters. PSD-95 is located in the postsynaptic region and is the main skeleton protein. Changes in the expression level of SYP can affect the release of presynaptic transmitters and synaptic plasticity, while PSD-95 is involved in the regulation of the structure and signal transmission of the postsynaptic membrane. SYP and PSD-95 play different roles in the process of synaptic plasticity and participate in the formation of learning and memory by regulating signal transmission and structural changes in the pre- and postsynaptic regions. The present invention detected the effects of combined fluoroaluminum exposure on the mRNA and protein expression of SYP and PSD-95, proteins related to synaptic plasticity in the hippocampus of second-generation offspring mice.

[0045] (1) Effects on the mRNA expression of SYP and PSD-95 The results of the effects of combined fluoride and aluminum exposure on the expression of SYP and PSD-95 mRNA in the hippocampus of the second-generation rats are as follows Figure 1As shown, compared with the control group, the mRNA expression of PSD-95 in the hippocampus of the second-generation offspring of each poisoning group was decreased; except for the fluorine-aluminum combined group, the mRNA expression levels of SYP in the other poisoning groups were reduced, and the differences were statistically significant.

[0046] (2) Effects on protein expression of SYP and PSD-95 The results of the effects of combined fluoride and aluminum exposure on the expression of SYP and PSD-95 proteins in the hippocampus of the second-generation rats are as follows Figure 2 As shown, compared with the control group, the expression of SYP and PSD-95 proteins in the hippocampus of the second-generation offspring mice in each exposure group was downregulated, and the difference was statistically significant.

[0047] 2. Determination of the effect of combined fluoride and aluminum exposure on genome-wide DNA methylation 5-methylcytosine (5-mC) is the most abundant methylation modification in mammalian genomic DNA. It can precisely regulate the gene expression of key biological processes and participate in the occurrence and development of various neurodegenerative diseases by affecting neuronal survival and synaptic plasticity. The present invention uses ELISA to detect the whole genome DNA methylation level of the second generation hippocampal tissue after fluorine-aluminum combined exposure.

[0048] The results are as follows Figure 3 As shown, compared with the control group, the whole genome DNA methylation level in the hippocampus tissue of the second-generation offspring of each exposed group was reduced, and the difference was statistically significant.

[0049] 3. Determination of the effects of combined fluoride and aluminum exposure on the expression levels of DNMT1, DNMT3a, and DNMT3b The process of DNA methylation regulating the development and differentiation of nerve cells is accomplished through the cooperation between DNMT1, which maintains methylation, and DNMT3a and DNMT3b, which are responsible for de novo methylation. DNMTs are involved in the dynamic regulation of DNA methylation. Abnormal changes in expression levels during the methylation process lead to learning and memory problems in animals, degenerative changes in the nervous system, and low methylation levels of brain neurons. Abnormal DNA methylation modification affects neuronal differentiation and survival, causing lesions in the nervous system.

[0050] In order to clarify the mechanism by which the combination of fluorine and aluminum leads to abnormal changes in methylation levels, the changes in the levels of methyltransferases DNMT1, DNMT3a and DNMT3b were further detected.

[0051] (1) Effects on DNMT1, DNMT3a, and DNMT3b mRNA expressions The results of the effects of combined fluoride and aluminum exposure on the mRNA expression of DNMT1, DNMT3a, and DNMT3b in the hippocampus of the second-generation rats are as follows Figure 4As shown, compared with the control group, the expression level of DNMT1 mRNA in the hippocampus tissue of the fluoride group and the (fluoride + aluminum) group was decreased; the expression of DNMT3a and DNMT3b mRNA in the hippocampus tissue of each poisoning group was decreased, and the difference was statistically significant.

[0052] (2) Effects on DNMT1, DNMT3a and DNMT3b protein expression The results of the effects of combined fluoride and aluminum exposure on the expression of DNMT1, DNMT3a and DNMT3b proteins in the hippocampus of the second-generation offspring mice are as follows Figure 5 As shown in the figure, compared with the control group, the protein expression levels of DNMT1, DNMT3a and DNMT3b in the hippocampal tissue of each exposure group were reduced, and the difference was statistically significant.

[0053] Therefore, the reduction of genomic methylation levels is one of the mechanisms by which combined exposure to fluorine and aluminum leads to damage of hippocampal synaptic plasticity.

[0054] Example 2: Effect of folic acid on synaptic plasticity damage induced by combined exposure to fluoride and aluminum 1. Experimental Methods 1. Establishment of NG108-15 cell model of fluoride-aluminum combined with folic acid intervention (1) Cell recovery and culture NG108-15 cells are a type of mouse neuroma cells. The cryopreservation tubes containing NG108-15 cells (from the Shanghai ATCC cell bank) were taken out of liquid nitrogen and immediately placed in a 37°C water bath until the cells were completely thawed. The cells were transferred into a 15 mL centrifuge tube using a pipette, and a certain amount of culture medium was added to make the total volume about 4 mL. The tubes were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 5 mL of culture medium was added, and the mixture was transferred to a culture flask. The culture flask was placed at 37°C and 5% CO. 2 Culture in a constant temperature incubator and change the medium in time for subculture.

[0055] (2) Determination of cell viability NG108-15 cells in good growth state in logarithmic growth phase were selected and cultured at a density of 1.0×10 5 After 100 μL / well of cell suspension was inoculated in a 96-well plate and cultured for 24 h, the original culture medium in the well was aspirated, and 100 μL of different concentrations of toxic substances were added to each well for 24 h (6 replicates were set for each group); 10 μL of CCK-8 working solution was added to each well of the plate, and after culturing at 37 °C for 1 h, the absorbance (OD value) at 450 nm was measured using an enzyme reader. Cell viability = [(absorbance of the experimental group - absorbance of the blank group) / (absorbance of the control group - absorbance of the blank group)] × 100%.

[0056] (3) Cell processing and grouping Determination of the concentration of fluorine and aluminum combined poisoning: According to the previous research of the research group, NaF and AlCl 3 IC 50 80 and 320 mg / L respectively, choose 1 / 2 of the IC 50 The dose was used as the toxic concentration in this experiment, so the toxic dose of NaF was 40 mg / L (1 / 2 IC 50 ), AlCl 3 The toxic dose is 160 mg / L (1 / 2 IC 50 ).

[0057] Determination of the effect of fluoride and aluminum on the viability of NG108-15 cells: The density was 1.0×10 5 100 μL / well of the cell suspension was inoculated into a 96-well plate and cultured for 24 h. The original culture medium in the well was aspirated and 100 μL of 40 mg / L NaF + 0 mg / L AlCl was added to each well. 3 、0mg / LNaF+160mg / LAlCl 3 、40mg / LNaF+160mg / LAlCl 3 The cells were cultured in the culture medium for 24 h (each group had 6 replicate wells); 10 μL of CCK-8 reagent was added to each well, and after culture at 37 °C for 1 h, the absorbance (OD value) at 450 nm was measured using an enzyme reader to calculate the cell viability.

[0058] Determination of folic acid intervention concentration: Preparation of folic acid mother solution: weigh 10 mg of folic acid and dissolve it in 10 mL of culture medium under light-proof conditions, filter and distribute it with a 0.22 μm sterile filter to prepare a mother solution with a concentration of 1000 μg / mL and store it in a -20 ℃ refrigerator for use (keep away from light); dilute it with culture medium to different concentrations before use.

[0059] Determination of the effect of folic acid on the viability of NG108-15 cells: The density was 1.0×10 5 100 μL / well of the cell suspension was inoculated into a 96-well plate and cultured for 24 h. The original culture medium in the well was aspirated and 100 μL of culture medium containing 4 mg / L, 8 mg / L, 16 mg / L and 32 mg / L folic acid was added to each well (6 replicate wells in each group) and cultured for 24 h. 10 μL of CCK-8 working solution was added to each well. After culturing at 37 °C for 1 h, the OD value at 450 nm was measured using an enzyme reader to calculate the cell viability.

[0060] Experimental groups: According to the results of the effects of different concentrations of folic acid on cell viability, it was found that after 24 hours of treatment with 16 mg / L folic acid on NG108-15 cells, the cell survival rate was high and the intervention effect was obvious, so the treatment concentration of folic acid was selected as 16 mg / L. The specific groups are shown in Table 3.

[0061] Table 3 Experimental groups Six-well plate cell plating and treatment: Take cells in the logarithmic growth phase and inoculate them in a 6-well plate. After 80% of the cells have grown attached to the wall, pour out the culture medium, wash with PBS three times, and suck out the remaining liquid in the air with a pipette. According to the above determined infection dose, add the prepared NaF and AlCl 3 The culture medium was added to the six-well plate, 2 mL / well, and exposed for 24 hours to establish the NG108-15 cell model of fluorine-aluminum combined exposure. After 24 hours of exposure to the above NG108-15 cells, the folic acid intervention group was replaced with a culture medium containing 16 mg / L folic acid, and the cells were collected after culturing for another 24 hours for subsequent experiments.

[0062] 2. ELISA detection of DNA methylation level in the whole genome of NG108-15 cells (1) Extraction of cell genomic DNA After the infection and intervention, the cells were treated with trypsin digestion solution to form a cell suspension, collected in a 1.5 mL EP tube, washed twice with PBS, and then centrifuged at 10,000 rpm for 1 min, and the supernatant was poured out. Then, the cell genomic DNA was extracted according to the instructions of the genomic DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.).

[0063] (2) Detection of whole genome DNA methylation levels The whole genome DNA methylation level of cells was detected according to the instructions of the 5-mC DNA ELISA kit (purchased from ZYMO RESEARCH, USA), and the 5-mC content was determined using an enzyme reader.

[0064] 3. qRT-PCR detection of SYP, PSD-95, DNMT1, DNMT3a and DNMT3b mRNA expression (1) Total RNA extraction After the infection and intervention, the cells were washed three times with pre-cooled PBS and then discarded. The remaining liquid in the six-well plate was sucked out with a pipette, and 1 mL of Trizol was added to each well to blow the cells and mix them. The cell suspension was transferred to a 1.5 mL centrifuge tube and allowed to stand for 10 min. RNA was then extracted according to the instructions of Trizol Reagent RNA extraction reagent (purchased from Invitrogen, USA) and stored in a -80°C refrigerator for later use. The RNA concentration was determined using an enzyme marker.

[0065] (2) Reverse transcription of total RNA into cDNA The steps of reverse transcribing total RNA into cDNA are the same as those in Example 1.

[0066] (3) qRT-PCR detection of related gene expression levels in cells ① Primer synthesis: SYP, PSD-95, DNMT1, DNMT3a, DNMT3b and GAPDH (internal reference) primers were designed and synthesized by Shanghai Jierui Biotechnology Co., Ltd. The specific information is shown in Table 4.

[0067] Table 4 qRT-PCR primer sequence list ② The reaction system and reaction procedure of qRT-PCR are the same as those of qRT-PCR in Example 1.

[0068] 4. Western Blotting (WB) to detect the expression of SYP, PSD-95, DNMT1, DNMT3a and DNMT3b proteins After the infection and intervention, the cells were washed twice with pre-cooled PBS, and the remaining liquid in the wells was sucked out with a pipette. 110 μL of lysis solution containing 1% protease inhibitor (PMSF) was added to each well. The cells were scraped with a cell scraper and placed on ice for 1 h. The cell suspension was scraped until clear with a cell scraper and then pipetted into a 1.5 mL EP tube. The tube was centrifuged at 4 °C and 14,000 rpm for 15 min. The supernatant was taken and the prepared protein was quantified by the BCA method. PBS and 5×SDS loading buffer were added to adjust the sample to the same protein concentration. The sample was placed in a boiling water bath for 10 min and then stored at -80 °C for later use.

[0069] Protein electrophoresis was performed on SYP, PSD-95, DNMT1, DNMT3a and DNMT3b. After development with developer, the optical density values ​​were calculated using Image-J software and statistical analysis was performed.

[0070] 2. Experimental Results 1. Effect of folic acid on NG108-15 cell viability Compared with the control group, 4 mg / L, 8 mg / L, 16 mg / L and 32 mg / L folic acid treatment alone had no significant effect on the viability of NG108-15 cells, and the difference was not statistically significant ( p >0.05) (see Figure 6 ), the folic acid concentration with the best efficacy and no significant effect on cell viability, i.e. 16 mg / L folic acid, was selected as the intervention dose.

[0071] 2. Effect of folic acid on the viability of NG108-15 cells exposed to fluoride and aluminum Compared with the control group, the viability of NG108-15 cells in each exposure group was reduced, and the difference was statistically significant ( p <0.05). The cells were treated with 16 mg / L folic acid and different concentrations of toxic substances. It was found that folic acid could significantly improve the decrease in NG108-15 cell viability caused by fluorine-aluminum combined poisoning. Compared with the corresponding general poisoning group, the viability of NG108-15 cells in each folic acid treatment group increased, and the difference was statistically significant (see Figure 7 ).

[0072] 3. Effect of folic acid on the mRNA and protein expression levels of synaptic plasticity-related proteins SYP and PSD-95 in NG108-15 cells co-exposed to fluoride and aluminum (1) Effects on the mRNA expression of SYP and PSD-95 Compared with the control group, except for the folic acid group, the mRNA expression of SYP in each experimental group was decreased, and except for the (fluorine + folic acid) group and the (aluminum + folic acid) group, the expression of PSD-95 in the other experimental groups was decreased, and the difference was statistically significant ( p <0.05); compared with the corresponding general poisoning groups, the mRNA expression of SYP in each group after folic acid treatment increased, and the mRNA expression of PSD-95 in the (fluorine + folic acid) group and the (aluminum + folic acid) group increased, and the difference was statistically significant ( p <0.05) (see Figure 8 ).

[0073] (2) Effects on protein expression of SYP and PSD-95 Compared with the control group, except for the folic acid and (fluorine + folic acid) groups, the protein expression of SYP in the other experimental groups decreased, and the protein expression of PSD-95 in all groups decreased, with statistically significant differences ( p <0.05). Compared with the corresponding general poisoning group, after folic acid treatment, the protein expressions of SYP and PSD-95 in each group of cells increased, and the difference was statistically significant ( p <0.05) (see Fig. 9 ).

[0074] 4. Effect of folic acid on the whole genome DNA methylation level of NG108-15 cells exposed to fluoride and aluminum Compared with the control group, the whole genome DNA methylation levels in the fluoride group, aluminum group, and (fluoride + aluminum) group were decreased, and the differences were statistically significant ( p <0.05). Compared with the corresponding general exposure groups, after folic acid treatment, the whole genome DNA methylation level in the (aluminum + folic acid) group and the (fluorine + aluminum + folic acid) group increased, and the difference was statistically significant ( p <0.05) (see Fig.10 ).

[0075] 5. Effects of folic acid on DNMT1, DNMT3a and DNMT3b in NG108-15 cells exposed to fluoride and aluminum (1) Effects on DNMT1, DNMT3a, and DNMT3b mRNA expressions Compared with the control group, except for the folic acid group, the expression of DNMT1 and DNMT3a mRNA in the cells of each group decreased; except for the (fluorine + aluminum + folic acid) group, the expression of DNMT3b mRNA in the cells of the other experimental groups decreased, and the difference was statistically significant. Compared with the corresponding general poisoning group, the expression of DNMT1 and DNMT3a mRNA in the cells of each group increased after folic acid treatment; in the (aluminum + folic acid) group, the expression of DNMT3b mRNA increased, and the difference was statistically significant ( p <0.05) (see Fig.11 ).

[0076] (2) Effects on DNMT1, DNMT3a and DNMT3b protein expression Compared with the control group, except for the folic acid group, the protein expression levels of DNMT1, DNMT3a and DNMT3b in the cells of the other experimental groups were all decreased, and the difference was statistically significant. Compared with the corresponding general poisoning groups, after folic acid treatment, the protein expression level of DNMT1 in the cells of the (fluorine + folic acid) group and the (fluorine + aluminum + folic acid) group increased, and the protein expression levels of DNMT3a and DNMT3b in the cells of the experimental groups increased, and the difference was statistically significant ( p <0.05) (see Fig.12 ).

[0077] In summary, the reduction of genomic methylation level is one of the mechanisms of hippocampal synaptic plasticity damage and learning and memory damage caused by fluorine-aluminum combined exposure. The mRNA and protein expressions of SYP and PSD-95 were reduced in the fluorine-aluminum combined poisoning group; while the 5-mC level in the cells was reduced, the mRNA and protein expression levels of DNMT1, DNMT3a and DNMT3b in each group were reduced. After the intervention of folic acid in the present invention, compared with the corresponding fluorine-aluminum combined poisoning group, the mRNA and protein expressions of SYP and PSD-95 in each group were increased, the 5-mC level in the cells was increased, and the mRNA and protein expression levels of DNMT1, DNMT3a and DNMT3b were increased, indicating that folic acid can improve the synaptic plasticity damage caused by fluorine-aluminum combined by increasing the genomic DNA methylation level.

[0078] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0079] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these changes and variations.

Claims

1. Application of folic acid in the preparation of drugs for improving learning and memory impairment caused by abnormal synaptic plasticity.

2. The use according to claim 1, characterized in that: The folic acid is used to prepare a drug for improving learning and memory impairment caused by abnormal synaptic plasticity due to combined exposure to fluorine and aluminum.

3. The use according to claim 1, characterized in that: The drug contains folic acid as the only active ingredient.

4. The use according to claim 1, characterized in that: The drug further comprises pharmaceutically acceptable excipients and / or carriers.

5. The use according to claim 1, characterized in that: The dosage form of the drug includes tablets, powders, granules or injections.

6. The use according to claim 1, characterized in that: The auxiliary material is any one or more of a filler, a disintegrant, a lubricant, a suspending agent, a binder, a sweetener, a flavoring agent, a preservative, a matrix and an antioxidant.

7. The use according to claim 1, characterized in that: The injection is a solution or an emulsion.

8. The use according to claim 1, characterized in that: The folic acid is used for preparing an expression promoter of synaptic protein SYP or PSD-95.

Images