CN113599540A - Application of miRNA-195-5p in inhibiting or reducing nerve cell injury caused by maltol aluminum - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and provides application of miRNA-195-5p in inhibiting or reducing nerve cell damage caused by maltol aluminum. The miRNA-195-5p overexpression is applied to inhibiting or reducing nerve cell damage caused by maltol aluminum. PC12 cells were used, using Al (mal)₃Constructing a tau protein hyperphosphorylation model, interfering cells by miRNA-195 overexpression, detecting the expression of miRNA-195 by qT-PCR, and detecting the expression conditions of ERK, P-ERK, tau5, PHF (tau-Ser396) and NFT (tau-Ser202, Thr205) by Western Blot. Laboratory clues are provided to reveal the molecular mechanism of hyperphosphorylation of aluminum-induced tau proteins.

Description

Application of miRNA-195-5p in inhibiting or reducing nerve cell injury caused by maltol aluminum

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of miRNA-195-5p in inhibition or reduction of nerve cell injury caused by maltol aluminum.

Background

The widespread use of aluminum makes it readily accessible to humans, and access to such metals includes air, food, and water. However, the physiological role of aluminum in the body is not currently known, but numerous studies have shown that excessive aluminum exposure can cause damage to various organs or systems of the body. With more aluminum toxicity studies focusing on aluminum toxicity studies on nerve cells, many scholars found that excessive aluminum exposure can cause nerve cell damage and even death, leading to loss of nerve cell function, with damage being more severe with increasing aluminum exposure doses. Among them, it was found that toxicity of aluminum to nerve cells is mainly achieved by hyperphosphorylating tau protein.

Tau protein is mainly present in the nervous system and is a neuronal Microtubule-associated protein (MAP), which is mainly distributed in neurons of the frontal, temporal, hippocampal and entorhinal regions of the brain. The physiological functions of tau protein in normal brain are to bind with tubulin to promote its polymerization to form microtubules, maintain microtubule stability, reduce dissociation of tubulin molecules, and induce microtubule bundling, thereby participating in the construction of neurons and the conduction of nerve signals. Under physiological conditions, tau protein functions at a level of phosphorylation of about 30 residues, most of which are threonine (pThr) or serine (pSer) sites, and thus tau protein is under a hypophosphorylated state in the normal body. Under pathological conditions, tau undergoes abnormal post-translational modifications, increasing the phosphorylation of tau 3-4 fold compared to normal brain, and this hyperphosphorylated tau has a significantly reduced ability to bind microtubules, leading to microtubule disassembly. Tau proteins dissociated from microtubules are more susceptible to misfolding and aggregation by some aggregation inducing agents, are aggregated into pathogenic double helical filaments (PHFs), and finally are mixed with Straight Filaments (SF) to form neurofibrillary tangles (NFTs), leading to neuronal toxicity and cognitive disorders.

It has been reported that hyperphosphorylation of tau protein can even lead to neuronal cell death. In addition, studies have shown that tau phosphorylation aggregation is inhibited, and cognitive impairment and neuronal damage are improved. The above studies suggest that tau protein is closely associated with neurotoxicity.

An imbalance in the regulation of the protein kinase and phosphatase systems is a direct cause of abnormal phosphorylation of Tau protein. Several protein kinases are known to catalyze phosphorylation of Tau protein, among which extracellular regulated protein kinases (ERK), which are key enzymes in the regulation of Tau protein phosphorylation levels, belong to a member of the MAPK pathway and are serine/threonine protein kinases. MEK acts as an upstream protein of ERK, activating ERK by phosphorylation of two regulatory sites Tyr 204/187 and Thr 202/185.

ERK has very important functions in regulating the proliferation and differentiation of neuron, raising learning and memory, etc. Over-activation of ERK has been shown to induce a variety of diseases including cancer, inflammation, developmental disorders, and neurological disorders, among others. Activated ERK can regulate the phosphorylation of microtubule-associated proteins in the cytoplasm and maintain the cellular morphology and cytoskeleton homeostasis.

miRNA is a non-coding small molecule RNA containing 21-24 nucleotides and having the function of regulating gene expression at the translation level or the post-transcription level. The pre-miRNAs are exported from the nucleus and further processed in the cytoplasm via a complex comprising Dicer and transactivation response RNA binding protein (TRBP) to form mature miRNAs. Mature miRNAs function in the RNA-induced silencing complex (RISC), which is either fully complementary to the target gene 3'UTR sequence, degrading its mRNA, or incompletely complementary to the target gene 3' UTR sequence, inhibiting its translation, i.e. miRNA affects protein synthesis by inhibiting translation or inducing degradation of target mRNAs.

mirnas are widely found in the nervous system, with some mirnas being specific or rich for specific brain regions. They act as key regulators of diverse biological functions, including synaptic plasticity and neurogenesis, and they can indirectly affect neurogenesis by regulating the proliferation and self-renewal of neural stem cells.

The miRNA-15 family comprises miRNA-15, miRNA-16, miRNA-195 and miRNA-497, and the down-regulation of the expression of the miRNA-15 family is a main mechanism for activating ERK to cause abnormal phosphorylation of tau protein in a Dicer knockout model. Since mirnas of the same family have the same seed region and can recognize the same mRNA segment, miRNA-15, miRNA-16, miRNA-195, and miRNA-497 can recognize the same mRNA segment, but different mirnas of the same family act at different levels and different stages. It has been found that miRNA-195 levels in the brain decrease as the disease progresses from normal aging to early AD, and that cerebrospinal fluid (CSF) miRNA-195 levels in MCI subjects correlate positively with cognitive performance measured by a mini-mental state examination (MMSE) and negatively with CSF tau levels. The researchers knock out endogenous miRNA-195 through a lentiviral vector, and find that rat dementia can be induced.

Disclosure of Invention

The invention provides application of miRNA-195-5p in inhibiting or reducing nerve cell damage caused by maltol aluminum. PC12 cells were used, using Al (mal)₃Constructing a tau protein hyperphosphorylation model, interfering cells by miRNA-195 overexpression, detecting the expression of miRNA-195 by RT-PCR, and detecting the expression conditions of ERK, P-ERK, tau5, PHF (tau-Ser396) and NFT (tau-Ser202, Thr205) by Western Blot. Laboratory clues are provided to reveal the molecular mechanism of hyperphosphorylation of aluminum-induced tau proteins.

The invention is realized by the following technical scheme: the application of miRNA-195-5p in inhibiting or reducing nerve cell injury caused by maltol aluminum and the application of miRNA-195-5p overexpression in inhibiting or reducing nerve cell injury caused by maltol aluminum.

The miRNA-195-5p is overexpressed to inhibit ERK activation, reduce tau protein phosphorylation and inhibit or reduce nerve cell damage caused by maltol aluminum.

The miRNA-195-5p is over-expressed for 36h to inhibit or reduce nerve cell damage caused by maltol aluminum.

The nucleotide sequence of the miRNA-195-5p is shown as SEQ ID NO. 1.

The miRNA-195-5p upstream primer is as follows: 5'-CCTAGCAGCACAGAA A-3', respectively; the downstream primer is: 5'-GAGCAGGCTGGAGAA-3' are provided.

The present invention employs PC12 cell, a tumor cell of the sympathetic nervous system. It has good and stable growth performance and is suitable for gene transfection research. In addition, the cell membrane of PC12 has a Nerve Growth Factor (NGF) receptor, and it recognizes the NGF, grows a neurite, and differentiates into a cell having sympathetic neuron properties. According to the invention, aluminum maltol is used for contamination, and miRNA screening finds that, compared with a control group, miRNA-195-5P is down-regulated in the aluminum-contaminated group, and the difference has statistical significance (P is less than 0.05). In addition, through the establishment of low, medium and high aluminum staining cell models, the expression level of miRNA-195-5P is gradually reduced along with the increase of aluminum staining concentration, and the difference has statistical significance (P < 0.05). The results show that the maltol aluminum can inhibit the expression of miRNA-195-5p of PC12 cells.

The invention successfully adopts a functional acquisition strategy to determine the role of miRNA-195 in hyperphosphorylation of aluminum-induced tau protein. Using PC12 cells, the experiments were divided into blank control group, blank plasmid group, 200. mu. mol/L Al (mal)₃Group, miRNA195 mics + 200. mu. mol/L Al (mal)₃And (4) grouping. By using cause analysis, the result shows that the total phosphorylated tau protein, PHF and NFT expression of the miRNA195mimics group is reduced compared with the control group, and the result indicates that the expression of miRNA-195-5p can inhibit hyperphosphorylation of tau protein and 200 mu mol/L Al (mal)₃The expression levels of total phosphorylated tau protein, PHF and NFT are obviously increased, which indicates that maltol aluminum can cause tau protein abnormal phosphorylation. Simultaneous displaymiRNA-195-5p and Al (mal)₃Interaction exists among tau5, PHF and NFT expressions, and the miRNA-195-5p is involved in Al (mal)₃Resulting in abnormal phosphorylation of tau protein.

After miRNA-195-5P is over-expressed, mRNA-ERK and total ERK protein in cells are not changed, and only the expression level of P-ERK is reduced, so that miRNA-195-5P influences tau protein phosphorylation by inhibiting ERK activation and does not directly act on mRNA-ERK and total ERK. Immunofluorescence analysis shows that through miRNA-195-5p overexpression intervention, the fluorescence density of tau5 protein in cytoplasm is reduced, and PHF and NFT fluorescence plaques are reduced; under a light microscope, cell damage is reduced, axons become longer and cell contact is increased, which indicates that the over-expression of miRNA-195-5p can save the cell damage caused by aluminum. Taken together, hyperphosphorylation of tau protein by aluminum in PC12 cells may be effected by inhibiting miRNA-195 expression, leading to increased ERK activation.

In conclusion, Al (mal)₃The contamination can inhibit the expression of miRNA-195-5p of PC12 cells, so that ERK is abnormally activated, and tau protein is hyperphosphorylated.

Compared with the prior art, the results of the invention show that: al (mal)₃The contamination can inhibit the expression of miRNA-195-5p of PC12 cells, so that ERK is abnormally activated, and tau protein is hyperphosphorylated. Therefore, the miRNA-195-5p is intervened or the ERK activation is inhibited to effectively improve the cytotoxicity caused by the maltol aluminum, and a scientific basis is provided for preventing and treating the cognitive function damage.

Drawings

FIG. 1 shows different Al (mal)₃Performing concentration treatment on an intracellular aluminum ion fluorescence signal;

FIG. 2 shows different Al (mal)₃Cell viability after concentration treatment, in the figure: the difference being statistically significant for different letter representations, P<0.05；

FIG. 3 shows different Al (mal)₃Cell morphology after concentration treatment;

FIG. 4 shows different Al (mal)₃The expression level of miRNA-195-5p after concentration processing; in the figure: the difference being statistically significant for different letter representations, P<0.05；

FIG. 5 shows different Al (mal)₃The expression level of mRNA-ERK after concentration treatment, the difference of letters in the figure indicates that the difference has statistical significance P<0.05；

FIG. 6 shows different Al (mal)₃Relative expression amount of P-ERK protein after concentration treatment; the difference in the letters in the figure is statistically significant P<0.05；

FIG. 7 shows different Al (mal)₃Relative expression of tau protein, PHF protein, NFT protein after concentration treatment, the difference in letters in the figure representing the statistical significance P<0.05；

FIG. 8 shows different Al (mal)₃Fluorescence signal of tau5 in PC12 cells after concentration treatment;

FIG. 9 shows different Al (mal)₃The concentration of PHF fluorescence signal in PC12 cell;

FIG. 10 shows different Al (mal)₃The fluorescence signal of NFT in PC12 cells after concentration treatment;

FIG. 11 shows the expression level of miRNA-195-5p of PC12 cells at different time points when miRNA-195-5p is overexpressed; the letters in the figure are different and indicate that the difference has statistical significance P < 0.05;

FIG. 12 is a morphogram of PC12 cells after miRNA-195-5p overexpression;

FIG. 13 shows the expression level of miRNA-195-5P after the overexpression of miRNA-195-5P in PC12 cells and the difference of letters in the figure indicates that the difference has statistical significance P < 0.05;

FIG. 14 shows the expression level of mRNA-ERK after overexpression of miRNA-195-5p in PC12 cells;

FIG. 15 shows the expression of P-ERK protein after overexpression of miRNA-195-5P in PC12 cells; the letters in the figure are different and indicate that the difference has statistical significance P < 0.05;

FIG. 16 shows the expression levels of tau5, PHF and NFT proteins after the overexpression of miRNA-195-5P in PC12 cells, wherein the difference in letters indicates that P is less than 0.05;

FIG. 17 shows the fluorescent signal of tau5 in PC12 cells after overexpression of miRNA-195-5 p;

FIG. 18 shows the fluorescent signal of PHF in PC12 cells after overexpression of miRNA-195-5 p;

FIG. 19 shows the fluorescent signal of NFT in PC12 cells after overexpression of miRNA-195-5 p.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and the disclosures and references cited herein and the materials to which they refer are incorporated by reference.

Those skilled in the art will recognize that many equivalents to the specific embodiments of the invention described, which are obvious from routine experimentation, are intended to be encompassed by the present application.

The experimental procedures in the following examples are conventional unless otherwise specified. The instruments used in the following examples are, unless otherwise specified, laboratory-standard instruments; the experimental materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

Example (b): materials for experiments

1. Experimental cell lines: rat adrenal pheochromocytoma cells (PC 12). Is from the cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences.

2. Experimental reagent: see table 1.

Table 1: reagents and sources for the assay

3. An experimental instrument: see table 2.

Table 2: instrument for experiment

Name of instrument	Manufacturer of the product
		CO2Constant temperature incubator	Thermo Fisher, USA
SW-CJ-2F type superclean bench	Antai air technology, Inc., China
		Milli-Q Reference ultra-pure water machine	Congress instruments & meters science & technology Limited, China
Low-speed centrifuge	Anhui Zhongkejia scientific instruments, China
		5424R low temperature ultracentrifuge	Eppendorf, Germany
Modle enzyme mark instrument	Bio-Rad, USA
		Horizontal shaking table	Beijing, Hex instruments factory, China
DYY-7C electrophoresis apparatus	Beijing, Hex instruments factory, China
		Membrane rotating instrument	Bio-Rad, USA
Gel imager Universal Hood II	Bio-Rad, USA
		PCR instrument	Roche diagnostics products Ltd, Switzerland

4. Solution preparation

(1) Aluminum maltol (Al (mal)₃) Solution: 0.1931g of AlCl₃·6H₂Dissolving O in 40mL of high-pressure double distilled water to prepare 20mmol/L AlCl₃A solution; 0.3024g of maltol was dissolved in 40mL of double distilled water which was passed through under high pressure to prepare a 60mmol/L maltol solution. Filtering with 0.22 μm filter membrane, and subpackaging, and storing at-20 deg.C when in use₃Mixing with maltol solution in equal volume to obtain 10mmol/L Al (mal)₃The pH of the solution is adjusted to 7.4 by 10 percent NaOH solution and the solution is prepared for use.

(2) Complete medium: DMEM high-sugar culture medium containing 10% fetal calf serum and 1% streptomycin mixed solution is mixed uniformly and stored at 4 ℃.

(3) Acry: Bis (30:0.8) 30%: 60g of acrylamide and 1.6g of methylene bisacrylamide are dissolved in a small amount of double distilled water, the volume is adjusted to 200ml, the solution is filtered by 0.45 mu m filter paper, and the solution is stored in a brown bottle at 4 ℃.

(4) Tris. Cl/SDS, 4 × (pH 8.8): weighing 18.2g of Tris, dissolving in a small amount of double distilled water, adjusting the pH to 8.8, adding the double distilled water to a constant volume of 100ml, finally adding 0.8g of SDS, performing ultrasonic dissolution, and storing at room temperature.

(5) Tris. Cl/SDS, 4 × (pH 6.8): weighing 6.05g of Tris, dissolving in a small amount of double distilled water, adjusting the pH value to 6.8, adding the double distilled water to a constant volume of 100ml, finally adding 0.8g of SDS, performing ultrasonic dissolution, and storing at room temperature.

(6) 10% ammonium persulfate solution: 0.4g of ammonium persulfate is weighed and dissolved in 4ml of double distilled water, and the mixture is uniformly mixed and stored at 4 ℃ for one week.

(7)10 times of film transfer liquid: 6g of Tris and 28.8g of glycine are dissolved in double distilled water, and the volume of the solution is up to 1L by adding the double distilled water.

(8)1 × membrane transfer liquid: take 10X 100mL of the membrane transfer solution, add 200mL of methanol, and finally add 700mL of double distilled water to make the final volume 1L.

(9)4 × electrophoretic fluid: weighing 24.2g of Tris and 87.66g of glycine, adding a small amount of double distilled water to fully dissolve the Tris and the glycine, then adding the double distilled water to the total volume of 1L, and finally adding 2g of SDS to fully dissolve the Tris and the glycine.

(10)1 × electrophoretic fluid: the electrophoresis solution was diluted 4 times.

(11)10 × TBS solution: weighing Tris 12.13g and NaCl 8.006g, adding a small amount of distilled water to fully dissolve, adjusting pH to 7.6, and then adding water to the total volume of 1L.

(12)1 × TBST: 10 × TBS was diluted 10 times, and 0.5mL of Tween 20 was added to 1L.

(13) Sealing liquid: 5g BSA or skimmed milk was weighed and added to 100mL 1 XTSST, and the mixture was dissolved sufficiently and ready for use. Pho S396 Ser202, Thr 205.

(14) Cell protein extract: mixing phosphatase inhibitor, protease inhibitor and RAPI lysate at a ratio of 1:1: 98.

(15) miRNA-195-5p mim and NC mimic stock solutions: 250 mu L of high-pressure double distilled water is added into 5nmol of mim freeze-dried powder to prepare 20 mu mol/L of mim stock solution.

(16)8-HQ stock solution: 0.7258g of 8-HQ powder was weighed out, and 10mL of DMSO was added to give a stock solution concentration of 5X 10⁵μmol/L。

Second, Experimental methods

1. Cell culture

Rat adrenal medulla pheochromocytoma cell (PC12) with a volume fraction of 5% CO at 37 deg.C₂Subculturing in a constant-temperature cell culture box. When the cells are recovered, taking out the frozen cells, thawing the frozen cells in a constant-temperature water bath at 37 ℃ within 3min, transferring the thawed cell suspension into a 15ml centrifugal tube, centrifuging for 5min at 1000rpm, discarding supernatant, adding 1ml culture solution, and beating by blowingMixing, transferring cell suspension into culture flask containing 3mL complete culture solution, mixing in shape of 8, placing at 37 deg.C and 5% CO by volume fraction₂Culturing in a constant-temperature cell culture box.

Cell passage: firstly, absorbing and discarding original culture solution, adding 3ml of D-hank's for cleaning for 2 times, and absorbing and discarding; then, 1mL trypsin-EDTA digestive fluid is added, the digestion is carried out for 1min at 37 ℃, 2mL complete culture fluid is used for stopping the digestion, all the fluid in the culture bottle at the moment is transferred to a 15mL centrifuge tube, the centrifuge is carried out for 5min at 1000rpm, the supernatant is sucked off, 4mL complete culture fluid is added into the cell sediment, the cell sediment is blown and uniformly mixed, 1mL of each cell suspension is respectively transferred to 4 culture bottles containing 3mL complete culture fluid, the cell suspension is uniformly mixed in a shape of a Chinese character '8', and the mixture is placed in a 37 ℃ culture bottle with 5% volume fraction CO₂Culturing in a constant-temperature cell culture box.

Collecting cells: collecting cell sediment (same as ' cell passage '), washing 1mL of D-hank's for 3 times, finally transferring the cell sediment into a 1.5mL EP tube, centrifuging at 1000rpm for 5min, discarding supernatant, and placing the collected cell sediment in a refrigerator at-80 ℃ for standby.

Freezing and storing cells: selecting cells in logarithmic phase, collecting cell sediment (same as cell passage), mixing complete culture medium, fetal calf serum and DMSO in a freezing medium according to a ratio of 4:5:1, adding 1mL of freezing medium into each bottle of cell sediment, blowing, beating, mixing uniformly, transferring into a 1.5mL freezing tube, freezing for 1h at-20 ℃ for-80 ℃ for a long time at 4 ℃ for 30min, and recovering the cells once every three months.

2. Cell viability assay

The cell pellet was collected (as in "cell passage"), 1mL of complete medium was added, the mixture was blown and mixed, and 0.5mL of the cell suspension was used for cell viability assay. To 0.5mL of the cell suspension, a certain amount of complete culture medium was added and inoculated into a 96-well plate. In a 96-well plate, 7 sets of six duplicate wells of 200. mu.L cell suspension per well were set up at a concentration gradient of 0. mu. mol/L, 100. mu. mol/L, 200. mu. mol/L, 300. mu. mol/L, 400. mu. mol/L, 500. mu. mol/L, 600. mu. mol/L Al (Al) (mal)3 concentration. When the cells have grown to the logarithmic growth phase, Al (mal)3 is added in the concentration gradient described above and the incubator is kept at 37 ℃ for 24 h. After 24 hours, the original culture solution in the 96-well plate was discarded, 110. mu.L (100. mu.L of complete culture solution and 10. mu.L of CCK8 solution) of the complete culture solution and CCK-8 mixed solution was added to each well, and six wells containing only the culture solution and CCK-8 mixed solution without cells were added, and placed in a 37 ℃ incubator for 1 hour, and absorbance measurement was carried out with a microplate reader at a wavelength of 450 nm.

Cell survival rate ═ [ (As-Ab)/(Ac-Ab) ] × 100%, where: as: experimental well (cell-containing medium, CCK-8, test substance), Ac: control wells (medium with cells, CCK-8, no test substance), Ab: blank wells (medium without cells and test substance, CCK-8).

3. Cell contamination and grouping

Al(mal)₃The group of toxicants was a control group, 100. mu. mol/LAl (mal)₃Group, 200. mu. mol/LAl (mal)₃Group, 400. mu. mol/LAl (mal)₃And (4) grouping. Six well plates were first rinsed with culture medium. Then two bottles of cells with the cell density of 90% are selected, cell precipitates are collected (same as passage), a certain amount of complete culture medium is added, blown, uniformly mixed and inoculated into 4 six-well plates, each hole is 1.5mL, and cross mixing is carried out. When the cells grow to logarithmic phase, 0. mu.L, 15. mu.L, 30. mu.L, 60. mu.L of Al (mal) was added to each well₃In each hole, Al (mal)₃The concentrations are respectively 0 mu mol/L, 100 mu mol/L, 200 mu mol/L and 400 mu mol/L, three compound wells with the same dosage are infected with the virus for 24 hours, and then the sample is collected.

4. Cell transfection and grouping

Divided into control group, empty plasmid group, 200. mu. mol/LAl (mal)₃Group, miRNA-195-5p overexpression group, namely miRNA-195-5p mimics group, miRNA-195-5p mimics +200 mu mol/LAl (mal)₃And (4) grouping. Transfection was performed when cells were grown to 70% in six-well plates. And taking 17.5 mu L of miRNA-195-5p mimic stock solution to be put into a 1.5ml EP tube A, adding 82.5 mu L of DMEM, and uniformly mixing. Taking 10 mu L of NC mimic stock solution to be put into a 1.5ml EP tube B, adding 90 mu L of DMEM, and mixing uniformly. mu.L and 24. mu.L of Lipofectamine 2000 were placed in 1.5ml of EP tube C and tube D, respectively, 58. mu.L of LDMEN was added to tube C, and 76. mu.L of DMEM was added to tube D, and the mixture was mixed well. Mixing tube A and tube C, and tube B and tube D respectively to a final volume of 200 μ L, respectively blowing and mixing, and standing at room temperature for 15 min. Original culture in the pipette well plateLiquid, blank control and 200. mu. mol/LAl (mal)₃Adding 1mL of DMEM culture solution without serum and double antibody into each hole of the group; adding 1950 mu L of DMEM and 50 mu L of mixed liquid of a tube B and a tube D into each hole of the NC mimcs group, and uniformly mixing; group of miRNA-195-5p mimics and miRNA-195-5pmimics + 200. mu. mol/LAl (mal)₃Add 71.4. mu.L DMEM and 28.6. mu.L of the mixture of tube A and tube C to each well of each group, mix them well, 3 multiple wells of each group, and culture them for 6 h. After 6h, the culture medium in the wells was changed to 1.5mL of complete culture medium containing serum and diabody, and the culture was continued for 18 h. After 18h, 200. mu. mol/LAl (mal)₃Group and miRNA-195-5pmimics + 200. mu. mol/LAl (mal)₃30 μ L of Al (mal) was added to each well of the group₃Mixing the solutions, culturing for 24 hr, and collecting.

5. Total RNA extraction

(1) Preparing consumables: pressurizing 1mL of the gun tip, 200 muL of the gun tip, 20 muL of the gun tip, 1.5mL of the EP tube, 4mL of the EP tube and double distilled water in advance, drying the gun tip and the EP tube for later use, and cooling the double distilled water and placing the double distilled water at 4 ℃ for later use. Trizon, chloroform, isopropanol and absolute ethyl alcohol for later use.

(2) And adding 1mL of Trizon into the cell sediment, blowing, uniformly mixing, and standing at room temperature for 5min to completely separate the protein nucleic acid complex.

(3) Chloroform was added continuously, 0.2mL of chloroform was added per 1mL of Trizon, the tube cap was closed, shaken vigorously for 15s, and left at room temperature for 2-3 min.

(4) Centrifugation at 12000rpm for 15min at 4 ℃ divided the sample into three year layers: the red organic phase, the middle and upper colorless aqueous phases, with RNA predominantly in the aqueous phase, were transferred to a new, high-pressure 1.5mL EP tube.

(5) Adding equal volume of isopropanol into the water phase solution, mixing by inversion, and standing at room temperature for 10 min.

(6) Centrifuge at 12000rpm for 10min at 4 ℃ and carefully discard the supernatant.

(7) Adding 75% ethanol (prepared with anhydrous ethanol and RNase-free water) into the obtained precipitate, covering the tube cover, flicking the tube wall with finger, and washing the precipitate. 1mL of 75% ethanol was added per 1mL of Trizon used.

(8) Centrifuge at 12000rpm for 3min at 4 ℃ and carefully discard the supernatant.

(9) Repeating the steps (7) and (8) for 2-3 times.

(10) Standing the precipitate at room temperature for 2-3min, air drying, adding 30-100 μ L RNase-free water, flicking the tube wall, dissolving RNA sufficiently, centrifuging instantaneously, and storing in-80 deg.C refrigerator for use.

6. Cell protein extraction

Preparing protein extract from phosphatase inhibitor, protease inhibitor and RAPI lysate according to the ratio of 1:1: 98. Collecting the collected cell precipitate, adding 60-90 μ L protein extract into each group of cell precipitate (6 wells), blowing, mixing, and incubating on ice for 20min to fully lyse the cells. Centrifuging at 12000rpm for 10min, taking the supernatant in a new 1.5mL EP tube, marking, the supernatant is the extracted cell protein.

7. BCA protein quantification

(1) BSA (bovine serum albumin) standard is shown in Table 3

Table 3:

pipe number	Diluent amount (double distilled water, μ L)	BSA Standard solution (. mu.L)	BSA Standard solution Final concentration (μ g/. mu.L)
				A	0	100	2
B	100	100	1
				C	100	100(B tube)	0.5
D	100	100(C tube)	0.25
				E	100	100(D tube)	0.125
F	100	100(E tube)	0.0625
				G	100	0	0 (blank control)

(2) BCA working solution

And mixing the BCA-A solution and the BCA-B solution according to the ratio of 50:1 to prepare the BCA working solution.

The amount of the working solution is (the number of BSA standard products plus the number of samples) multiplied by the number of multiple wells multiplied by the volume of the working solution to be added into each well.

(3) The protein sample to be tested is diluted by 25 times, 4 mu L of protein stock solution and 96 mu L of double distilled water.

(4) The standard and diluted protein samples were added to a 96-well plate at 25 μ L each, in 3 duplicate wells each.

(5) Adding 200 μ LBCA working solution into each well, mixing well, incubating at 37 deg.C for 30min, cooling to room temperature, and completing detection within 3-5 min.

(6) The absorbance of each sample and BSA standard was measured by a microplate reader, lambda 540-590 nm.

(7) And drawing a standard curve, calculating the relationship between the protein concentration and the absorbance by using the absorbance of the standard substance and the known concentration, and calculating the concentration of the protein to be detected according to the absorbance of the protein to be detected.

(8) And (3) diluting by using the protein extract, and adjusting the concentrations of all proteins to be detected to be consistent with the lowest concentration group. Then 5 × loading buffer was added to each protein sample, 5 × loading buffer: protein ═ 1: 4.

(9) Boiling in boiling water, adding protein sample into boiling water, boiling for 5min, cooling to room temperature, and standing at-20 deg.C.

8. Western Blot for detecting expression level of related protein

(1) Cleaning and assembling the glass plate: the newly purchased glass plate was soaked in detergent water for one to two days. Cleaning both sides with tap water, cleaning with distilled water, placing on a shelf, and air drying in a drying oven for use. And aligning and clamping the clean glass plate in the clamping groove to prepare glue injection.

(2) 10% of separation glue and lamination glue are prepared, and the formula is shown in table 4.

Table 4:

solutions of	10% separation gel	Lamination adhesive
			Acry:Bis(30:0.8)30％	5.00mL	1.30mL
Tris·Cl/SDS，pH 8.8(1.5mM)	3.75mL	—
			Tris·Cl/SDS，pH 6.8(1.0mM)	—	2.50mL
Double distilled water	6.25mL	6.10mL
			10% ammonium persulfate solution	100μL	100μL
TEMED	20μL	20μL

(3) Injecting glue: the separation gel was injected from one side of the glass plate to two thirds of the height of the glass plate. And then continuously adding 2mL of isopropanol in a fast-scratching and slow-adding manner, flattening the glue surface, standing for 25min, and pouring out the isopropanol after the separation and gelation are good. Adding the lamination glue continuously in the same way as the separation glue, and filling. The glue injection speed is controlled, and bubbles are prevented from being generated. Inserting the comb into the separation gel, taking care not to generate bubbles, standing for 25min, and slowly pulling out the comb after the separation gel is good. After the gel is prepared, the gel and the glass plate are installed on an electrophoresis tank, and after electrophoresis buffer solution is added, the gel starts to be ready for sample loading.

(4) Loading: taking out the protein sample, melting at room temperature, shaking, mixing uniformly, and centrifuging instantaneously. At first, 2 μ L and 4 μ L of maker are added to the head and the tail of the sample hole, and equal volume of protein sample is added to the sample hole in the middle.

(5) Electrophoresis: setting the voltage to be 60V, carrying out electrophoresis for 2.5h, stopping electrophoresis when bromophenol blue just runs out, and turning the membrane as soon as possible to prevent protein diffusion.

(6) Film transfer: 0.45 μm PVDF membrane was soaked in methanol. Firstly, taking out the membrane transferring clamp, and soaking the sponge pad and the filter paper by using the recovered membrane transferring liquid. Then a spongy cushion, filter paper, glue, a PVDF film, filter paper and a spongy cushion are sequentially placed on a blackboard of the film rotating clamp to clamp the film rotating clamp. Note that the PVDF membrane should be placed on the protein sites of the corresponding molecular weight and no air bubbles should be generated between each layer. Placing the clips into a film rotating groove, wherein the positive and negative poles correspond to each other, placing ice bags on two sides of the film rotating groove, pouring film rotating liquid, and setting the current at 300mA for 70 min.

(7) And (3) sealing: blocking solutions were prepared with 1 × TBST and either milk or BSA. And (4) after the film transfer is finished, taking out the PVDF film, and soaking the PVDF film in the sealing liquid as soon as possible to avoid drying the PVDF film. Blocking for 3h on a shaker at room temperature.

(8) Incubating the primary antibody: primary antibody was diluted with 1% skim milk or BSA at the specified ratio and each protein band was incubated with 2mL antibody dilution overnight at 4 ℃.

(9) Incubation of secondary antibody: the strips were washed 3 times with 1 × TBST for 10min each time. The secondary antibody was diluted with 1% skim milk or BSA at the stated ratio, and each protein band was incubated with 2mL of antibody dilution for 2h at 37 ℃.

(10) And (3) developing: the strips were washed 3 times with 1 × TBST for 10min each time. And preparing 200 mu L of luminous liquid for each protein band, developing by using a Universal Hood II imager, and finally analyzing the gray value by using Quantity One software.

9. Gene expression level detection

(1) Reverse transcription of miRNA-195-5p and internal reference U6

The total RNA concentration extracted was first determined and the volume of 1. mu.g of RNA was calculated. Reverse transcription kit using Bomby at 50. mu.L of 20. mu. mol/L Bulge-Loop^TMAdding miRNA RT Primer into 150 mu L of RNase-free water to prepare 5 mu mol/L Bulge-Loop^TMmiRNA RT Primer. The experiments were carried out according to the system of table 5 (on ice):

table 5:

mixing the above systems, and performing instant centrifugation, wherein the RT reaction procedure is 60min at 42 ℃ and 10min at 70 ℃.

(2) mRNA-ERK1, mRNA-ERK2, and mRNA-GA reverse transcription

The total RNA concentration extracted was first checked and a volume of 500ng RNA was calculated. Experiments were performed using the reverse transcription kit of TaKaRa according to the system of table 6 (prepared on ice):

table 6:

reagent	10 μ L system	Final concentration
			5×PrimerScript RT Master Mix(Pertect Real Time)	2μL	1×
Total RNA	*
			RNase-Free dH2O	To 10 μ L

Reaction system can be scaled up according to the requirement, and the 10 uL reaction system can use 500ng of Total RNA at maximum.

The above systems are mixed evenly and then are centrifuged instantly, and the RT reaction procedure is 15min at 37 ℃, 5s at 85 ℃ and 4 ℃.

(3) miRNA-195-5p, U6, mRNA-ERK, and mRNA-GA qt-PCR

The experiments were carried out according to the system of table 7 (on ice):

table 7:

reagent	20 μ L system	Final concentration
			2×SYBR Green Mix	10μL		1×
RT Product	2μL
			Forward Primer(5μmol/L)	0.8μL	200nmol/L
Reverse Primer(5μmol/L)	0.8μL	200nmol/L
			RNase-Free dH2O	To 20 μ L

The qPCR reaction procedure is shown in table 8:

table 8:

primer sequences are shown in table 9:

table 9:

10. cells Al (mal)₃And (3) fluorescence distribution detection: and (3) paving the cells on a laser confocal micropore dish, staining aluminum when the cells grow to 30%, and continuously culturing for 24 hours.

(1) The culture medium in the dish was aspirated and washed 3 times with 1% PBS for 5min each.

(2) 4% paraformaldehyde is fixed for 20min at normal temperature, and each dish is 1 mL. Then washed 3 times with 1% PBS for 5min each.

(3) 0.5% Triton (100% Triton diluted 200-fold in 1% PBS) was perforated on ice for 15min, 1mL per dish. Then washed 3 times with 1% PBS for 5min each.

(4) Taking 8-HQ stock solution, diluting with DMSO by 100 times to prepare 8-HQ working solution (500 mu mol/L) for aluminum staining, wherein each dish is 1mL, placing the dish in a cassette, and incubating for 30min at 37 ℃. Then washed 3 times with 1% PBS for 5min each. The subsequent steps should be carried out with care to avoid light.

(5) 1mg/mL DAPI was diluted to 5. mu.g/mL with 1% PBS, and nuclei were stained with 5. mu.g/mL DAPI, 1mL per dish, and incubated at room temperature for 10 min. Then washed 3 times with 1% PBS for 5min each.

(6) 1mL of 1% PBS was added to each dish and detected by confocal laser detection.

11. Tau protein immunofluorescence: and (3) paving the cells on a laser confocal micropore dish, and staining aluminum or intervening when the cells grow to 30%.

(1) The same as (1), (2) and (3) of 10.

(2) Appropriate amount of BSA powder was weighed, 5% BSA was prepared with 1% PBS, 1mL per dish, and CDC was searched for blocking at room temperature for 1 h.

(3) 5% BSA was aspirated off and primary antibody diluted with 1% BSA was added at 500. mu.L per dish overnight at 4 ℃.

(4) Washing with 1% PBS for 5min 3 times. A fluorescent secondary antibody diluted with 1% BSA was added, placed in a cassette, and incubated at 37 ℃ for 1 h. The subsequent steps should be carried out with care to avoid light.

(5) Washing with 1% PBS for 5min 3 times.

(6) The same as (5) and (6) of 10.

Thirdly, statistical analysis: statistical analysis was performed using SPSS 22.0 software. All data are measured data, adopt

And (4) showing. Single-factor analysis of variance is adopted for comparison among multiple groups; and (4) pairwise comparison: the variance is tested by LSD and the variance is tested by Dunnett. Analysis of P-ERK and Al (mal) by analysis of the causes₃Alone and in interaction. The test level α is 0.05 (both sides).

Fourthly, the result

1. Experimental results of different doses of aluminum maltol contamination

A. Distribution of maltol aluminum in PC12 cells after maltol aluminum treatment: detection of intracellular Al (mal) by confocal laser₃Distribution of (2). The results are shown in FIG. 1, and the fluorescence signal shows that aluminum is mainly distributed in cytoplasm and concentrated around nucleus, and along with Al (mal)₃The fluorescence signal gradually increases with increasing concentration.

B. Effect of maltol aluminum on the viability and morphology of PC12 cells: with different concentrations of Al (mal)₃Cells (0, 100. mu. mol/L, 200. mu. mol/L, 300. mu. mol/L, 400. mu. mol/L, 500. mu. mol/L, 600. mu. mol/L) were treated and 24h later the cell viability was determined using the CCK8 kit. The result of the detection is shown in figure 2,as can be seen from fig. 2: 0. 100. mu. mol/L, 200. mu. mol/L, 300. mu. mol/L, 400. mu. mol/L, 500. mu. mol/L, 600. mu. mol/L Al (mal)₃The cell viability after the infection is 1, 92.05, 87.69, 85.62, 82.88, 78.67 and 71.75 respectively. Wherein, the ratio of Al (mal) is 100 mu mol/L, 200 mu mol/L and 400 mu mol/L₃Since the cell viability was maintained at 80% or more in the concentration treatment, 100. mu. mol/L, 200. mu. mol/L, and 400. mu. mol/L of Al (mal) were selected₃The concentrations were used as low, medium and high dose groups.

By 100. mu. mol/L, 200. mu. mol/L, 400. mu. mol/LAl (mal)₃After the treatment, the cell morphology was observed by a 100 Xinverted microscope, and the cell morphology was 100. mu. mol/LAl (mal) as shown in FIG. 3, as compared with the control group₃There was a slight, but not significant, reduction in the group cells; 200 μmol/LAl (mal)₃The cell density of the group is reduced, the cells are flat, the stereoscopic impression is reduced, the axon is shortened, and the cell connection is reduced; 400 μmol/LAl (mal)₃The cell density of the group is obviously reduced, the cells are flat, the axons are broken, the cell connection is reduced, the cell bodies are reduced, and the cell nucleus is enlarged.

C. Effect of Maltophenolate on expression of miRNA-195-5p in PC12 cells

And detecting the expression quantity of different groups of miRNA-195-5p by adopting qT-PCR. The results are shown in Table 11 and FIG. 4, and show that the expression level of miRNA-195-5p is reduced along with the increase of the aluminum staining dose. 100 μ M Al (mal) compared to control group₃Group, 200. mu.M Al (mal)₃Group, 400. mu.M Al (mal)₃The groups all have statistical differences and are respectively reduced by 14.7 percent, 28.0 percent and 40.9 percent (P)<0.05). With 100. mu.M Al (mal)₃Group comparison, 200. mu.M Al (mal)₃And 400. mu.M Al (mal)₃The expression level of group miRNA-195-5P is obviously reduced (P)<0.05)。

Table 11: expression level of miRNA-195-5p after treatment of different Al (mal)3 concentrations

Group of	Amount of expression
		Control group	1.000±0.000a
100μM Al(mal)3	0.853±0.037b
		200μM Al(mal)3	0.720±0.027c
400μM Al(mal)3	0.591±0.064c

D. Effect of maltol aluminium on expression of mRNA-ERK, total ERK and P-ERK proteins of PC12 cells

And detecting the expression quantity of different groups of mRNA-ERK by adopting qT-PCR. The results are shown in FIG. 5, which shows that the results follow Al (mal)₃The concentration is increased, and the expression quantity of mRNA-ERK is not changed.

And detecting the relative expression quantity of the total ERK and P-ERK proteins by using Western blot. The results are shown in Table 12, FIG. 5, and FIG. 6, and show that the results follow Al (mal)₃The total ERK protein expression quantity is not changed when the concentration is increased, and the P-ERK protein expression quantity is gradually increased. 100 μ M Al (mal) compared to control group₃No significant difference of the expression level of the P-ERK protein (P) in the group>0.05)；200μMAl(mal)₃The expression level of the group P-ERK protein is increased by 1.736 times (P)<0.05)；400μM Al(mal)₃The expression level of the group P-ERK protein is increased by 1.993 times (P)<0.05)。

Table 12: expression level of mRNA-ERK after treatment with different Al (mal)3 concentrations

Group of	Amount of expression
		Control group	1.000±0.000a
100μM Al(mal)3	0.984±0.140a
		200μM Al(mal)3	0.976±0.083a
400μM Al(mal)3	1.035±0.100a

Note: the letters differ to indicate that the difference has a statistical significance P < 0.05.

E. Effect of Maltophenolate on expression of tau5, PHF and NFT proteins in PC12 cells

And detecting the relative expression amounts of tau, PHF and NFT proteins by using Western blot. The results are shown in FIG. 7, which shows that tau, PHF and NFT expression levels are increased with the increase of the aluminum-staining concentration. The Tau5 results showed 100. mu.M Al (mal) compared to the control group₃No significant difference in expression level of group tau protein (P)>0.05)，200μmol/LAl(mal)₃Group, 400. mu. mol/LAl (mal)₃The expression level of the histone is respectively increased by 1.220 times and 1.459 times (P)<0.05). The PHF protein results show 100. mu. mol/L Al (mal) compared to the control group₃Group, 200. mu. mol/L Al (mal)₃Group, 400. mu. mol/L Al (mal)₃The expression level of histone is respectively increased by 1.184 times, 1.533 times and 2.500 times (P)<0.05). NFT protein results show 100. mu.M Al (mal) compared to control₃No significant difference (P) occurs in the expression quantity of the NFT proteins in the group>0.05)，200μmol/L Al(mal)₃Group, 400. mu. mol/L Al (mal)₃The expression level of histone is increased by 1.394 times and 2.417 times respectively (P)<0.05)。

F. Effect of Maltophenolate on PC12 cell tau5, PHF, NFT fluorescence distribution

Immunofluorescence results are shown in FIGS. 8, 9, and 10, showing that tau5 is mainly localized to cytoplasm, PHF is mainly localized to nucleus, and NFT is distributed in both nucleus and cytoplasm, both with Al (mal)₃The increase in concentration gradually increased the fluorescence signal. By Al (mal)₃After treatment, both tau5 and NFT appeared as larger fluorescent plaques in the cytoplasm; in contrast, PHF shows the transfer of protein from nucleus to cytoplasm, and a weak fluorescent signal is observed in the cytoplasm of cells.

2. Results of miRNA-195-5p intervention experiment

A. Effect of miRNA-195-5p overexpression on morphology of PC12 cells

Firstly, different miRNA-195-5p overexpression time groups are set, qT-PCR results are shown in figure 11, and the results show that the expression quantity of miRNA-195-5p overexpression is the highest at 48h, so that the experiment setting selects miRNA-195-5p overexpression to intervene for 48h for breeding, and subsequent indexes are detected.

After miRNA-195-5p mimics treatment, cell morphology was observed by 100 × inverted microscope, and the results are shown in FIG. 12, which shows that Al (mal)₃The number of cells is gradually reduced by the infection, the axon is reduced and broken, the cell body becomes round, and after the treatment of miRNA-195-5p overexpression, the number of the cells is increased, the axon is lengthened, and the cell connection is increased.

B. Effect of miRNA-195-5p overexpression on expression of miRNA-195-5p in PC12 cells

And detecting the expression quantity of different groups of miRNA-195-5p by adopting qT-PCR. The results are shown in Table 13 and FIG. 13, and the results show that the miRNA-195-5P expression level of the blank plasmid group is unchanged and the miRNA-195-5P expression level of the miRNA-195-5P overexpression group is 1428.634 times higher (P is 1428.634 times higher) compared with the control group (P is a gene with a gene<0.05); with 200. mu. mol/L Al (mal)₃Group comparison, miRNA-195-5p overexpresses + 200. mu. mol/L Al (mal)₃The expression level of the group miRNA-195-5p is increased by 1826.305 times.

Table 13: expression level of miRNA-195-5p overexpression stem prognosis miRNA-195-5p

Group of	Amount of expression
		Control group	1.000±0.000a
200μM Al(mal)3	0.750±0.029b
		Empty plasmid group	1.066±0.138a
miRNA-195-5p overexpression group	1429.634±76.693c
		miRNA-195-5p overexpression + 200. mu.M Al (mal)3Group of	1370.479±146.804c

C. Effect of miRNA-195-5P overexpression on expression of mRNA-ERK, total ERK and P-ERK proteins of PC12 cells

And detecting the expression quantity of different groups of mRNA-ERK by adopting qT-PCR. The results are shown in Table 14 and FIG. 14, and show that the miRNA-195-5p overexpression does not cause the change of the expression level of the mRNA-ERK. NC mimcs group, 200. mu.M Al (mal) compared to control group₃Group, miRNA-195-5p mimics +200 mu mol/L Al (mal)₃No significant difference of mRNA-ERK expression quantity appears in the group.

Table 14: expression level of miRNA-195-5p overexpression stem prognosis mRNA-ERK

Note: the letters differ to indicate that the difference has a statistical significance P < 0.05.

The expression level of total ERK and P-ERK protein is detected by Westernon Blot. The results are shown in FIG. 15, which shows that the total ERK expression level is not changed among the groups; compared with the control group, the expression level of P-ERK in the empty leukocytic group is not changed, 200 mu mol/LAl (mal)₃The expression level of the P-ERK protein in the infected group is increased by 1.736 times (P)<0.05), the expression level of miRNA-195-5P over-expression group P-ERK protein is reduced by 16.69 percent (P)<0.05); analysis of the gene analysis shows that miRNA-195-5p is over-expressed and Al (mal)₃There is an interaction on P-ERK protein expression, namely miRNA-195-5P participates in Al (mal)₃Resulting in abnormal expression of P-ERK protein.

D. Effect of miRNA-195-5p overexpression on expression of tau, PHF and NFT proteins in PC12 cells

And detecting the relative expression amounts of tau5, PHF and NFT proteins by using Western blot. The results are shown in FIG. 16, which shows that the expression levels of tau5, PHF and NFT proteins in the blank plasmid group were unchanged compared with the control group, and that 200. mu. mol/L Al (mal)₃The expression levels of tau5, PHF and NFT in the infected group are respectively increased by 1.220 times, 1.533 times and 1.394 times (P)<0.05), the expression levels of tau5, PHF and NFT proteins of miRNA-195-5P overexpression groups are respectively reduced by 13.900%, 21.310% and 18.270% (P)<0.05); analysis of the gene analysis shows that miRNA-195-5p is over-expressed and Al (mal)₃Interaction on tau5, PHF, NFT protein expression, i.e., involvement of miRNA-195-5p in Al (mal)₃Tau5, PHF, NFT protein expression were abnormal due to infection.

E. Effect of miRNA-195-5p overexpression on PC12 cell tau5, PHF, NFT fluorescence distribution

Immunofluorescence results are shown in FIGS. 17, 18, and 19, and show that tau5 is mainly localized to cytoplasm, PHF is mainly localized to nucleus, and NFT is distributed in both nucleus and cytoplasm, both with Al (mal)₃The increase in concentration gradually increased the fluorescence signal. With 200. mu. mol/L Al (mal)₃Compared with the group, tau5, PHF and NFT are in miRNA-195-5p mics +200 mu mol/L Al (mal)₃The fluorescence signal of the group decreased and the fluorescent plaques decreased. Tau5 was mainly expressed as a decrease in overall fluorescence density following the dry prognosis of miRNA-195-5p mimics; PHF is mainly manifested by the disappearance of the fluorescent signal in the cytoplasm; NFT is mainly manifested as the dissipation of large fluorescent plaques in the cytoplasm.

The PC12 cell used in the invention is a tumor cell of sympathetic nervous system. It has good and stable growth performance and is suitable for gene transfection research. In addition, the cell membrane of PC12 has a Nerve Growth Factor (NGF) receptor, and it recognizes the NGF, grows a neurite, and differentiates into a cell having sympathetic neuron properties. Therefore, this study used the cells for modeling, and Al (mal)₃Cell contamination was performed and aluminum was detected by immunofluorescence to enter the cell and to be distributed mainly in the cytoplasm and axons.

Effect of Al on tau hyperphosphorylation: aluminum is ubiquitous in life, but is not essential to life, and is involved in biochemical reactions of the body, as a well-known neurotoxic metal, aluminum is accumulated in the hippocampal region, frontal cortex, etc. of the brain across the blood-brain barrier and placental barrier, and repeatedly shows accumulation in neuronal foci susceptible to cognitive impairment, and X-ray spectra demonstrate accumulation of aluminum in neurofibrillary tangles formed in the brain of patients with cognitive dysfunction. In addition, researchers have investigated the distribution of metals in the brain tissue of patients diagnosed with AD, and have shown that there is a deposition of aluminum in their brain tissue. This indicates that aluminum plays an important role in cognitive decline.

In the invention, a cell model for aluminum staining is constructed, and whether aluminum can enter cells is detected by fluorescent dye, the result shows that green fluorescent spots appear in the cells, and the fluorescent spots increase along with the increase of the concentration of the aluminum staining, so that the change of related molecules can be detected by establishing a model for aluminum accumulation in neurons by staining the cells with aluminum in vitro research.

The invention uses tau separately5 antibody, tau-pSer396 antibody and AT8(tau-pSer202, Thr205) antibody to examine total tau, PHF and NFT to investigate the expression of tau AT different phosphorylation sites. The results show that tau5 protein expression increases with increasing aluminum concentration, and PHF is administered at low doses (100. mu. mol/L Al (mal)₃) A clear increase was seen, whereas only a medium dose (200. mu. mol/L Al (mal)) of NFT was seen₃) A clear increase was observed, so we speculate that although aluminium may cause hyperphosphorylation of tau protein, PHF is more sensitive to aluminium toxicity and appears early, whereas NFT appears later. Furthermore, immunofluorescence revealed that total Tau represented by Tau5 antibody was detected predominantly in the cytoplasm, and that total Tau signal was enhanced after staining with aluminum, while its localization was unchanged. Antibodies against phosphorylated tau show the localization of tau in the nucleus. PHF is mainly localized in the nucleus, but it appears in the cytoplasm after aluminum staining, and nuclear signaling is also enhanced. NFT has a distribution in both nucleus and cytoplasm, but after staining with aluminum, it is mainly manifested by an increase in fluorescence signal in cytoplasm. Under a light microscope, axons of cells are obviously shortened after the cells are stained with aluminum, cell connection is reduced, and the cells are obviously reduced.

The above results all confirm that aluminum can cause abnormal phosphorylation of tau protein, leading to abnormal aggregation of tau protein, resulting in cytotoxicity. Thus, we propose that aluminium can cause hyperphosphorylation of tau protein, and that phosphorylation of tau protein by aluminium occurs primarily at the Ser396 site, with phosphorylation at the Ser202, Thr205 site occurring with increasing aluminium concentration.

Inhibition of ERK activation effects on Al-induced protein hyperphosphorylation: extracellular signal regulated kinases (ERKs) belong to the Mitogen Activated Protein Kinase (MAPK) family, play a role in signal cascades, and transmit Extracellular signals to intracellular targets.

The extracellular signal-regulated kinases, ERK, are largely classified into two, ERK1 and ERK2, are evolutionarily conserved, ubiquitous serine-threonine kinases that regulate cell signals under normal and pathological conditions. The phosphorylation sites of Tau protein are mainly threonine (pThr) or serine (pSer) sites, so we conclude that hyperphosphorylation of Tau protein by aluminum may be effected by activation of ERK.

In the present invention, first, Al (mal) is performed on cells₃Contamination, found 200. mu.M Al (mal) compared to control₃Group sum 400 μ M Al (mal)₃The expression level of the group P-ERK protein is respectively increased by 1.736 times and 1.993 times (P)<0.05). Furthermore, tau5 protein, PHF, and NFT were expressed at 200. mu.M Al (mal)₃Significant increase in groups occurred. Results Explanation, Al (mal)₃The effect of the toxicity caused the over-activation of ERK pathway is consistent with the abnormal phosphorylation of tau protein, suggesting that the over-activation of ERK pathway may be an upstream signal causing the over-phosphorylation of tau protein, and is also one of the reasons for the over-phosphorylation and cytotoxicity of tau in aluminum-induced PC12 cells.

And (4) conclusion: maltol aluminum can enter cells, mainly distributed in cytoplasm; tau5 protein is distributed predominantly in the cytoplasm; double helix filaments (PHF, p-Ser396) are distributed mainly in the nucleus; neurofibrillary tangles (NFT, p-Ser202, p-Thr205) are distributed both in the nucleus and in the cytoplasm. Furthermore, tau5 and NFT were expressed in cytoplasm after aluminum maltol exposure, and PHF was not only expressed in increased amounts but also transferred to cytoplasm.

Phosphorylation of tau by aluminum occurs primarily at the Ser396 site, with phosphorylation at the Ser202, Thr205 site occurring with increasing aluminum concentration.

Maltol aluminum can activate phosphorylated ERK resulting in abnormal phosphorylation of tau protein. Therefore, the cell cytotoxicity caused by maltol aluminum is effectively improved by inhibiting ERK activation, and a scientific basis is provided for preventing and treating cognitive function impairment.

miRNA-195-5p regulates the effect of hyperphosphorylation of aluminum-induced tau protein:

according to the invention, the miRNA of the cells of the aluminum staining group and the control group is screened by using the PC12 cells, and the miRNA-195-5p is down-regulated in the expression of the aluminum staining group, and the difference has statistical significance. In addition, through the establishment of low, medium and high aluminum staining cell models, the expression level of miRNA-195-5p is found to gradually decrease along with the increase of aluminum staining concentration, and the difference has statistical significance. The results indicate that aluminum in PC12 cells can affect the expression of miRNA-195-5 p.

We conclude that miRNA-195 can alleviate hyperphosphorylation of tau protein caused by aluminum and improve cognitive impairment by inhibiting protein kinase activation. The invention successfully adopts a functional acquisition strategy to determine the role of miRNA-195 in hyperphosphorylation of aluminum-induced tau protein. The results show that the expression levels of total phosphorylated tau protein, PHF and NFT in cells transfected with miRNA-195-5p mimics are all reduced, and the results show that the phosphorylation of tau protein is dependent on the expression of miRNA-195-5p, and suggest that the expression of miRNA-195-5p is inversely related to the phosphorylation degree of tau protein. Simultaneous miRNA-195-5p overexpression and 200. mu. mol/L Al (mal)₃Treatment, analysis of the causes showed that miRNA-195-5p was overexpressed and 200. mu. mol/L Al (mal)₃Interaction exists among tau5, PHF and NFT expressions, and the miRNA-195-5p is involved in Al (mal)₃Resulting in abnormal phosphorylation of tau protein.

After miRNA-195-5P is over-expressed, mRNA-ERK and total ERK protein in cells are not changed, and only the expression level of P-ERK is reduced, so that miRNA-195-5P influences tau protein phosphorylation by inhibiting ERK activation and does not directly act on mRNA-ERK and total ERK. Immunofluorescence analysis shows that by miRNA-195-5p overexpression intervention, tau5 protein in cytoplasm is reduced in fluorescence density, and PHF and NFT fluorescent plaques are reduced; under a light microscope, cell damage is reduced, axons become longer and cell contact is increased, which indicates that the over-expression of miRNA-195-5p can save the cell damage caused by aluminum. Taken together, hyperphosphorylation of tau protein by aluminum in PC12 cells may be effected by inhibiting miRNA-195 expression, leading to increased ERK activation.

In conclusion, Al (mal)₃The contamination can inhibit the expression of miRNA-195-5p of PC12 cells, so that ERK is abnormally activated, and tau protein is hyperphosphorylated.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Sequence listing

<110> university of Shanxi medical science

Application of <120> miRNA-195-5p in inhibiting or reducing nerve cell damage caused by maltol aluminum

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 87

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aacucuccug gcucuagcag cacagaaaua uuggcacggg uaagugaguc ugccaauauu 60

ggcugugcug cuccaggcag gguggug 87

Claims (5)

1. The application of miRNA-195-5p in inhibiting or reducing nerve cell damage caused by maltol aluminum is characterized in that: the miRNA-195-5p overexpression is applied to inhibiting or reducing nerve cell damage caused by maltol aluminum.
2. 2. Use according to claim 1, characterized in that: the miRNA-195-5p is overexpressed to inhibit ERK activation, reduce tau protein phosphorylation and inhibit or reduce nerve cell damage caused by maltol aluminum.
3. 3. Use according to claim 1, characterized in that: the miRNA-195-5p is over-expressed for 36h to inhibit or reduce nerve cell damage caused by maltol aluminum.
4. 4. Use according to claim 1, characterized in that: the nucleotide sequence of the miRNA-195-5p is shown as SEQ ID NO. 1.
5. 5. Use according to claim 1, characterized in that: the miRNA-195-5p upstream primer is as follows: 5'-CCTAGCAGCACAGAA A-3', respectively; the downstream primer is: 5'-GAGCAGGCTGGAGAA-3' are provided.

Images