CN113599540B - Application of miRNA-195-5p in preparation of reagent for inhibiting or reducing nerve cell damage caused by aluminum maltol - Google Patents

Abstract

The invention belongs to the technical field of medical biology, and provides an application of miRNA-195-5p in inhibiting or reducing nerve cell damage caused by aluminum maltol. The miRNA-195-5p overexpression is applied to inhibiting or reducing nerve cell damage caused by aluminum maltol. PC12 cells were used, using Al (mal) ₃ Constructing a tau protein hyperphosphorylation model, interfering cells by adopting miRNA-195 over-expression, detecting the expression of miRNA-195 by qT-PCR, and detecting the expression conditions of ERK, P-ERK, tau5, PHF (tau-Ser 396) and NFT (tau-Ser 202, thr 205) by Western Blot. Provides a laboratory clue for revealing the molecular mechanism of aluminium-induced tau protein hyperphosphorylation.

Description

Application of miRNA-195-5p in preparation of reagent for inhibiting or reducing nerve cell damage caused by aluminum maltol

Technical Field

The invention belongs to the technical field of medical biology, and particularly relates to application of miRNA-195-5p in preparation of a reagent for inhibiting or reducing nerve cell damage caused by aluminum maltol.

Background

The widespread use of aluminum has made it easy for humans to access aluminum, and the means of access to such metals include air, food, and water. However, the physiological role of aluminum in the body is currently unknown, but numerous studies have shown that excessive aluminum exposure can cause damage to multiple organs or systems of the body. More of these aluminum toxicity studies have focused on toxicity studies of aluminum on nerve cells, and many scholars have found that excessive aluminum exposure can cause nerve cell damage and even death, resulting in loss of nerve cell function, and that damage is more severe with increasing doses of aluminum exposure. Among these studies, aluminum toxicity to nerve cells was found to be achieved primarily by hyperphosphorylation of 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 lobe, temporal lobe, hippocampus and entorhinal region of the brain. The physiological function of tau protein in normal brain is to combine with tubulin to promote its polymerization to form microtubules, maintain microtubule stability, reduce dissociation of tubulin molecules, and induce microtubule bundling, thereby participating in neuron construction and nerve signal transmission. Under physiological conditions, tau protein functions at a phosphorylation level of about 30 residues, most of which are threonine (pThr) or serine (pSer) sites, so that in normal organisms tau protein is in a hypophosphorylated state. Under pathological conditions, tau protein undergoes abnormal post-translational modification, increasing the degree of phosphorylation of tau protein to 3-4 times that of normal brain, and this hyperphosphorylated tau protein has significantly reduced ability to bind microtubules, resulting in microtubule disintegration. Tau proteins dissociated from microtubules are more prone to misfolding and aggregation under the influence of some aggregation inducers, are polymerized into pathogenic duplex filaments (PHF), and finally mix with Straight Filaments (SF) to form neurofibrillary tangles (NFT), leading to neuronal toxicity and cognitive impairment.

Tau hyperphosphorylation has been reported to even lead to neuronal cell death. In addition, studies have found that tau protein phosphorylation aggregation is inhibited, and that both cognitive dysfunction and neuronal damage can be ameliorated. The above studies suggest that tau protein is closely related to neurotoxicity.

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

ERK plays a very important role in regulating proliferation and differentiation of neurons, improving learning and memory, and the like. Overactivation of ERK has been shown to induce a variety of diseases including cancer, inflammation, developmental disorders, and neurological diseases, among others. Activated ERK can regulate microtubule-associated protein phosphorylation in the cytoplasm, maintaining cell morphology and cytoskeletal homeostasis.

miRNA is a non-coding small molecule RNA, contains 21-24 nucleotides, and has the function of regulating gene expression at the level of translation or post-transcriptional level. The precursor miRNAs are exported from the nucleus and further processed in the cytoplasm by a complex, including Dicer and transactivation-response RNA binding protein (TRBP), to form mature miRNAs. Mature miRNAs play a role in RNA-induced silencing complexes (RISC), which are 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, that is, miRNAs affect protein synthesis by inhibiting translation or inducing degradation of the target mRNAs.

mirnas are widely found in the nervous system, some of which are specific or enriched for specific brain regions. They act as key mediators of diverse biological functions, including synaptic plasticity and neurogenesis, and they can indirectly affect neurogenesis by modulating 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 abnormal phosphorylation of tau protein of a Dicer knockout model caused by ERK. Mirnas of the same family have the same seed region and can recognize the same mRNA fragment, so that miRNA-15, miRNA-16, miRNA-195 and miRNA-497 can recognize the same mRNA fragment, but different miRNAs of the same family respectively act at different levels and at different stages. It has been found that the levels of miRNA-195 in the brain decrease as the disease progresses from normal aging to early AD, and that the levels of cerebrospinal fluid (CSF) miRNA-195 in MCI subjects are positively correlated with cognitive performance as measured by the simple mental state examination (MMSE), and negatively correlated with CSF tau levels. The learner knocks out endogenous miRNA-195 through a lentiviral vector, and the learner can induce rat dementia.

Disclosure of Invention

The invention provides an application of miRNA-195-5p in preparing a reagent for inhibiting or reducing nerve cell damage caused by aluminum maltol. PC12 cells were used, using Al (mal) ₃ Constructing a tau protein hyperphosphorylation model, interfering cells by adopting miRNA-195 over-expression, detecting the expression of miRNA-195 by RT-PCR, and detecting the expression conditions of ERK, P-ERK, tau5, PHF (tau-Ser 396) and NFT (tau-Ser 202, thr 205) by Western Blot. Provides a laboratory clue for revealing the molecular mechanism of aluminium-induced tau protein hyperphosphorylation.

The invention is realized by the following technical scheme: the application of miRNA-195-5p in preparing an agent for inhibiting or reducing nerve cell damage caused by aluminum maltol, wherein the miRNA-195-5p is over-expressed in the application for inhibiting or reducing the nerve cell damage caused by aluminum maltol.

The miRNA-195-5p overexpression inhibits ERK activation, reduces tau protein phosphorylation, and inhibits or reduces nerve cell damage caused by aluminum maltol.

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

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

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

The invention uses PC12 cells, which are tumor cells of the sympathetic nervous system. The method has good and stable growth performance, and is suitable for gene transfection research. Furthermore, the nerve growth factor (nerve growth factor, NGF) receptor exists on the cell membrane of PC12, and can recognize the growth factor (NGF), grow neurites, and differentiate into cells with sympathetic neuron characteristics. The invention uses maltol aluminum for contamination, and miRNA screening discovers that miRNA-195-5P is down-regulated in the expression of the aluminum-contaminated group compared with the control group, and the difference has statistical significance (P < 0.05). In addition, by establishing low, medium and high-dyeing aluminum cell models, the expression quantity of miRNA-195-5P is found to gradually decrease along with the increase of the concentration of the dyed aluminum, and the difference is statistically significant (P < 0.05). The results show that the aluminum maltol can inhibit the expression of the miRNA-195-5p of the 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 grouped into blank, and 200. Mu. Mol/LAl (mal) ₃ Group, miRNA195 micrometers group, miRNA195 micrometers+200. Mu. Mol/L Al (mal) ₃ A group. By using factorial analysis, the result shows that compared with a control group, the expression level of total phosphorylated tau protein, PHF and NFT of the miRNA195 micrometers group is reduced, and the result shows that the expression of miRNA-195-5p can inhibit tau protein hyperphosphorylation, and 200 mu mol/L Al (mal) ₃ The expression level of total phosphorylated tau protein, PHF and NFT is obviously increased, which indicates that abnormal phosphorylation of tau protein can be caused by aluminum maltol. miRNA-195-5p and Al (mal) are shown simultaneously ₃ Interaction with tau5, PHF, NFT expression indicates that miRNA-195-5p is involved in Al (mal) ₃ Resulting abnormal phosphorylation of tau protein.

After miRNA-195-5P is over expressed, mRNA-ERK and total ERK proteins in cells are not changed, and only the expression level of P-ERK is reduced, so that miRNA-195-5P can influence 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, we find that the fluorescence density of tau5 protein in cytoplasm is reduced, and PHF and NFT fluorescent plaques are reduced; under the light microscope, the cell damage is reduced, the axis mutation is long, and the cell contact is increased, which indicates that the cell damage caused by aluminum can be saved by the overexpression of miRNA-195-5 p. In conclusion, aluminium-induced tau hyperphosphorylation in PC12 cells may be exerted by inhibiting miRNA-195 expression, resulting in increased ERK activation.

To sum up, al (mal) ₃ Contamination can inhibit the expression of PC12 cell miRNA-195-5p, leading to aberrant activation of ERK, leading to tau protein hyperphosphorylation.

Compared with the prior art, the result of the invention shows that: al (mal) ₃ Contamination can inhibit the expression of PC12 cell miRNA-195-5p, leading to aberrant activation of ERK, leading to tau protein hyperphosphorylation. Therefore, the miRNA-195-5p is interfered or ERK activation is inhibited to effectively improve the cytotoxicity caused by the aluminum maltol, so that scientific basis is provided for preventing and treating the cognitive function injury.

Drawings

FIG. 1 shows different Al (mal) ₃ An intracellular aluminum ion fluorescence signal after concentration treatment;

FIG. 2 shows different Al (mal) ₃ Cell viability after concentration treatment, in the figure: the different letters represent differences with statistical significance, P<0.05；

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

FIG. 4 shows a different Al (mal) ₃ The expression level of miRNA-195-5p after concentration treatment; in the figure: the different letters represent differences with statistical significance, P<0.05；

FIG. 5 shows different Al (mal) ₃ The concentration of mRNA-ERK expression level after treatment, the letter differences in the figure indicate the difference with statistical significance P<0.05；

FIG. 6 shows different Al (mal) ₃ The relative expression level of the P-ERK protein after concentration treatment; the differences between letters in the figure are statistically significant P<0.05；

FIG. 7 shows different Al (mal) ₃ Relative expression levels of tau protein, PHF protein and NFT protein after concentration treatment, wherein differences in letters indicate statistically significant differences P <0.05；

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

FIG. 9 shows different Al (mal) ₃ Fluorescence signal of PHF in PC12 cells after concentration treatment;

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

FIG. 11 shows the expression levels of miRNA-195-5p in PC12 cells at different time points when miRNA-195-5p is overexpressed; the differences in the letters in the figure are statistically significant P <0.05;

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

FIG. 13 shows the expression level of miRNA-195-5P after the over-expression intervention of miRNA-195-5P of PC12 cells, wherein the difference between letters indicates that the difference is statistically significant P <0.05;

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

FIG. 15 is the expression of the P-ERK protein after the overexpression of miRNA-195-5P in PC12 cells; the differences in the letters in the figure are statistically significant P <0.05;

FIG. 16 shows the expression levels of tau5, PHF and NFT proteins after the interference of the overexpression of miRNA-195-5P of PC12 cells, wherein the difference between letters indicates that the difference is statistically significant P <0.05;

FIG. 17 shows fluorescence signals of tau5 in PC12 cells after overexpression of miRNA-195-5 p;

FIG. 18 shows fluorescence signals of PHF in PC12 cells after overexpression of miRNA-195-5 p;

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

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the 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, the disclosure of which is incorporated herein by reference as is commonly understood by reference.

Those skilled in the art will recognize that many equivalents to the specific embodiments of the invention described herein, as may be known by routine experimentation, are intended to be encompassed within the present application.

The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Examples: 1. materials used in experiments

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

2. Experimental reagent: see table 1.

Table 1: reagents for experiments and sources thereof

3. Experimental instrument: see table 2.

Table 2: instrument for experiment

Instrument name	Manufacturer' s
		CO 2 Constant temperature incubator	Thermo Fisher, U.S
SW-CJ-2F type ultra-clean bench	Antai air technologies Co., ltd., china
		Milli-Q Reference ultra-pure water machine	He Yu Instrument and meter science and technology Co., ltd, china
Low-speed centrifugal machine	Zhongjia scientific instruments Co., ltd., china in Anhui
		5424R low-temperature ultracentrifuge	Eppendorf, germany
Modle enzyme labeling instrument	Bio-Rad, U.S
		Horizontal shaking table	Beijing Liuyi Instrument factory, china
DYY-7C electrophoresis apparatus	Beijing Liuyi Instrument factory, china
		Film transferring instrument	Bio-Rad, U.S
Universal Hood II for gel imager	Bio-Rad, U.S
		PCR instrument	Roche diagnostics products, inc., switzerland

4. Solution configuration

(1) Aluminum maltol (Al (mal) ₃ ) Solution: 0.1931g AlCl ₃ ·6H ₂ O is dissolved in 40mL of double distilled water after high pressure to prepare 20mmol/L AlCl ₃ A solution; 0.3024g of maltol was dissolved in 40mL of double distilled water under high pressure to prepare a 60mmol/L maltol solution. Filtering with 0.22 μm filter membrane, packaging, and storing with-20, and when in use, alCl ₃ Mixing with maltol solution in equal volume to obtain 10mmol/L Al (mal) ₃ The solution was prepared by adjusting the pH to 7.4 with 10% NaOH solution.

(2) Complete medium: DMEM high sugar culture medium containing 10% fetal calf serum and 1% green streptomycin mixed solution, mixing, and storing at 4deg.C.

(3) Acry: bis (30:0.8) 30%: 60g of acrylamide and 1.6g of methylene bisacrylamide were dissolved in a small amount of double distilled water, the volume was fixed to 200ml, filtered through a 0.45 μm filter paper, and stored in a 4℃brown bottle.

(4) Tris-Cl/SDS, 4× (pH 8.8): weighing 18.2g of Tris, dissolving in a small amount of double distilled water, regulating the pH to 8.8, adding double distilled water to a volume of 100ml, finally adding 0.8g of SDS, dissolving by ultrasonic wave, and preserving 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, regulating the pH to 6.8, adding double distilled water to a volume of 100ml, finally adding 0.8g of SDS, dissolving by ultrasonic wave, and preserving at room temperature.

(6) 10% ammonium persulfate solution: weighing 0.4g of ammonium persulfate, dissolving in 4ml of double distilled water, uniformly mixing, preserving at 4 ℃ and keeping for one week.

(7) 10 x transfer solution: tris 6g and glycine 28.8g were dissolved in double distilled water and the double distilled water was added to a constant volume of 1L.

(8) 1 x transfer solution: 100mL of 10 Xtransfer membrane solution was taken, 200mL of methanol was added, and finally 700mL of double distilled water was added to give a final volume of 1L.

(9) 4 x electrophoretic fluid: 24.2g of Tris and 87.66g of glycine are weighed, a small amount of double distilled water is added to fully dissolve, then double distilled water is added to 1L of total volume, and finally 2g of SDS is added to fully dissolve.

(10) 1 x electrophoretic fluid: 4 Xthe electrophoresis solution was diluted 4-fold.

(11) 10 XTBS solution: tris 12.13g,NaCl 8.006g was weighed, added with a small amount of distilled water to dissolve thoroughly, pH was adjusted to 7.6, and then water was added to a total volume of 1L.

(12) 1 XTBST: 10 XTBS was diluted 10-fold and 0.5mL Tween 20 was added per 1L.

(13) Sealing liquid: 5g of BSA or skimmed milk was weighed, 100mL of 1 XTBST was added thereto, and the mixture was fully dissolved and prepared for use. PhosphoS 396 Ser202, thr205.

(14) Cell protein extract: preparing the phosphatase inhibitor, the protease inhibitor and the RAPI lysate into a mixed solution according to the ratio of 1:1:98.

(15) miRNA-195-5p mimc and NC mimc stock: 250. Mu.L of high-pressure double distilled water was added to 5nmol of mimc lyophilized powder to prepare 20. Mu. Mol/L of mimc stock solution.

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

2. Experimental method

1. Cell culture

Rat adrenal medullary pheochromocytoma cells (PC 12) at 37℃with a volume fraction of 5% CO ₂ Subculturing in a constant temperature cell incubator. When the cells are recovered, the frozen cells are taken out and melted in a constant temperature water bath kettle at 37 ℃ within 3min, the melted cell suspension is transferred into a 15mL centrifuge tube, the centrifugation is carried out at 1000rpm for 5min, the supernatant is discarded, 1mL of culture solution is added for blowing and mixing uniformly, the cell suspension is transferred into a culture bottle containing 3mL of complete culture solution, the 8-shaped culture solution is mixed uniformly, and the mixture is placed in a CO with the volume fraction of 5% at 37 DEG C ₂ Culturing in a constant temperature cell incubator.

Cell passage: firstly, absorbing and discarding the original culture solution, adding 3ml of D-hank's, cleaning for 2 times, and absorbing and discarding; then, 1mL of trypsin-EDTA digestion solution was added, digestion was stopped with 2mL of complete culture solution at 37℃for 1min, all the solution in the flask at this time was transferred to a 15mL centrifuge tube, centrifuged at 1000rpm for 5min, the supernatant was removed by aspiration, 4mL of complete culture solution was added to the cell pellet, and the mixture was blown and stirred uniformly, 1mL of each cell suspension was transferred to 4 flasks containing 3mL of complete culture solution, and the mixture was stirred uniformly in 8-letter, and placed at 37℃with a volume fraction of 5% CO ₂ Culturing in a constant temperature cell incubator.

Cell collection: cell pellet (same as "cell passage") was collected, 1mL of D-hank's was washed 3 times, and finally, cell pellet was transferred to 1.5mL of EP tube, centrifuged at 1000rpm for 5min, and the supernatant was discarded, and the collected cell pellet was placed in a-80℃refrigerator for use.

Cell cryopreservation: selecting cells in logarithmic growth phase, collecting cell sediment (same as 'cell passage'), mixing complete culture medium, fetal bovine serum and DMSO in the ratio of 4:5:1 in the frozen stock solution, adding 1mL of frozen stock solution into each bottle of cell sediment, blowing and mixing uniformly, transferring into a 1.5mL frozen stock tube, carrying out gradient freezing at 4 ℃, 30min, 20 ℃ for 1h, 80 ℃ for a long time, and recovering cells every three months.

2. Cell viability assay

Cell pellet (same as "cell passage") was collected, added to 1mL of complete medium, and mixed well by pipetting, 0.5mL of cell suspension was taken for cell viability assay. To 0.5mL of the cell suspension, a certain amount of the complete culture solution was added, and inoculated into a 96-well plate. In a 96-well plate, 7 groups of six duplicate wells each of 200. Mu.L of cell suspension were arranged at Al (mal) 3 concentration gradients 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 (mal) 3 was added in the above concentration gradient after the cells had grown to the logarithmic growth phase, and the mixture was placed in an incubator at 37℃for 24 hours. After 24 hours, the original culture solution in the 96-well plate was pipetted, 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 were added to each well, and at the same time, six wells containing only the culture solution and CCK-8 mixed solution without cells were added, and the wells were placed in a 37℃incubator for 1 hour, and absorbance was measured with an enzyme-labeled instrument at a wavelength of 450 nm.

Cell viability = [ (As-Ab)/(Ac-Ab) ]x100%, wherein: as: experiment holes (cell-containing Medium, CCK-8, test substance), ac: control wells (cell-containing medium, CCK-8, no test substance), ab: blank wells (medium without cells and test substance, CCK-8).

3. Cell contamination and grouping

Al(mal) ₃ The contamination group is control group, 100 mu mol/LAl (mal) ₃ Group, 200. Mu. Mol/LAl (mal) ₃ Group, 400. Mu. Mol/LAl (mal) ₃ A group. First, a six-well plate was rinsed with the culture solution. Then selecting two bottles of cells with the cell density reaching 90%, collecting cell sediment (same as 'passage'), adding a certain amount of complete culture medium, blowing and mixing uniformly, inoculating into 4 six-hole plates, and mixing uniformly in a cross way, wherein each hole is 1.5 mL. When the cells grew to the logarithmic phase, 0. Mu.L, 15. Mu.L, 30. Mu.L, 60. Mu.L of Al (mal) was added to each well, respectively ₃ Make Al (mal) in each hole ₃ The concentrations are respectively 0 mu mol/L, 100 mu mol/L, 200 mu mol/L and 400 mu mol/L, three compound holes are used at the same dose, and the sample is collected after 24 hours of contamination.

4. Cell transfection and grouping

Grouping into control group, empty white matter fraction group, 200. Mu. Mol/LAl (mal) ₃ Group miRNA-195-5p overexpression group, namely miRNA-195-5p micrometers group, miRNA-195-5p micrometers+200. Mu. Mol/LAl (mal) ₃ A group. Transfection was performed when cells grew to 70% in six well plates. 17.5. Mu.L of miRNA-195-5p mimic stock solution was taken in 1.5ml of EP tube A, 82.5. Mu.L of DMEM was added and mixed well. 10. Mu.L of NC mic stock solution was added to 1.5ml of EP tube B, and mixed well with 90. Mu.L of DMEM. 42. Mu.L and 24. Mu.L of the LLipofectamine 2000 were placed in 1.5ml of EP tube C and tube D, respectively, 58. Mu.L of DMEN was added to tube C, 76. Mu.L of DMEM was added to tube D, and the mixture was homogenized. Tube A and tube C were mixed separately, tube B and tube D were mixed, the final volumes were 200. Mu.L, and each were blown and mixed uniformly and left at room temperature for 15min. Original culture solution in the well plate was pipetted off, blank and 200. Mu. Mol/LAl (mal) ₃ 1mL of DMEM culture solution without serum and double antibodies is added to each well of the group; 1950 mu L of DMEM and 50 mu L of mixed liquid of the tube B and the tube D are added into each hole of the NC mimcs group, and the mixed liquid is uniformly mixed; miRNA-195-5p micrometers group and miRNA-195-5p micrometers+200. Mu. Mol/LAl (mal) ₃ 71.4 mu L of DMEM and 28.6 mu L of the mixed solution of tube A and tube C are added into each hole of each group, the mixed solution is uniformly mixed, and each group is cultured for 6 hours in 3 compound holes. After 6 hours, the culture solution in the hole is replaced to 1.5mL of complete culture solution containing serum and double antibodies, and the culture is continued for 18 hours. After 18h, 200. Mu. Mol/LAl (mal) ₃ Group and miRNA-195-5p micrometers+200. Mu. Mol/LAl (mal) ₃ Each well of the group was added with 30. Mu.L of Al (mal), respectively ₃ Mixing the solutions, continuously culturing for 24h, and then taking care.

5. Total RNA extraction

(1) Consumable preparation: the gun tip and the EP tube are dried for standby, and the double distilled water is cooled and then placed at 4 ℃ for standby. Trizon, chloroform, isopropanol and absolute ethanol are used for standby.

(2) 1mL of Trizon is added into the cell sediment, and the mixture is blown and mixed uniformly and left at room temperature for 5min, so that the protein accounting complex is completely separated.

(3) And adding chloroform continuously, adding 0.2mL of chloroform every 1mL of Trizon, covering a tube cover, shaking vigorously for 15s, and standing at room temperature for 2-3min.

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

(5) Adding equal volume of isopropanol into the aqueous phase solution, mixing the mixture evenly in a reverse way, and standing the mixture at room temperature for 10min.

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

(7) To the obtained precipitate, 75% ethanol (prepared with absolute ethanol and RNase-free water) was added, the tube cap was closed, the tube wall was flicked with a finger, and the precipitate was washed. 1mL of 75% ethanol was added per 1mL of Trizon.

(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 μl of RNase-free water, flicking the tube wall, dissolving RNA completely, centrifuging instantaneously, and storing in a refrigerator at-80deg.C.

6. Cellular protein extraction

Phosphatase inhibitor, protease inhibitor and RAPI lysate are mixed according to the following ratio of 1:1:98 to prepare protein extract. Collecting cell sediment, adding 60-90 μl of protein extract into each group of cell sediment (6 holes), blowing and mixing, and incubating on ice for 20min to allow the cells to be fully lysed. Centrifugation at 12000rpm for 10min gave the supernatant, which was the extracted cellular protein, in a fresh 1.5mL EP tube, labeled.

7. BCA protein quantification

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

Table 3:

(2) BCA working fluid

Mixing BCA-A solution and BCA-B solution at a ratio of 50:1 to obtain BCA working solution.

Working fluid usage= (number of BSA standard + number of samples) ×number of wells×volume of working fluid to be added per well.

(3) The protein sample to be tested was diluted 25-fold with 4. Mu.L of protein stock + 96. Mu.L of double distilled water.

(4) 25. Mu.L of each of the standard and diluted protein samples was added to each of 3 wells of a 96-well plate.

(5) 200 mu LBCA working solution is added into each hole, the mixture is fully and uniformly mixed, incubated for 30min at 37 ℃, cooled to room temperature and detected within 3-5 min.

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

(7) Drawing a standard curve, calculating the relation 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 concentration of all proteins to be detected to be consistent with the lowest concentration group. Then 5×loading buffer, was added to each group of protein samples: protein=1:4.

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

8. Western Blot detection of related protein expression levels

(1) Cleaning and assembling a glass plate: the newly purchased glass plate was immersed in the detergent water for one to two days. After both sides are scrubbed, the two sides are washed clean by tap water, then washed clean by distilled water, placed on a shelf and dried in a drying box for standby. And (5) aligning and clamping the clean glass plate in a clamping groove, and preparing for glue injection.

(2) 10% release gum and laminating gum were formulated and the formulation is shown in Table 4.

Table 4:

(3) And (3) glue injection: the release glue was injected from one side of the glass plate to the height of two thirds of the glass plate. And then continuously adding 2mL of isopropanol by adopting a rapid and slow adding mode, flattening the rubber surface, standing for 25min, and pouring the isopropanol after the separation and gelation are finished. And continuously adding laminating adhesive in the same way as the separating adhesive, and filling. And the glue injection speed is controlled, so that bubbles are avoided. Inserting the comb into the separating gel, taking no bubbles, standing for 25min, and slowly pulling out the comb after the gel is well separated. After the glue is prepared, the glue is mounted on an electrophoresis tank together with a glass plate, and after the electrophoresis buffer solution is added, the glue starts to be ready for loading.

(4) Loading: taking out the protein sample, melting at room temperature, shaking, mixing uniformly, and performing instantaneous centrifugation. Firstly, adding 2 mu L and 4 mu L of maker at the head and the tail of the sample hole, and sequentially adding an equal volume of protein sample into the middle sample hole.

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

(6) Transferring: a PVDF membrane of 0.45 μm was immersed in methanol. Firstly, taking out the film transferring clamp, and soaking the sponge cushion and the filter paper with recovered film transferring liquid. Then placing a sponge cushion, filter paper, glue, PVDF film, filter paper and sponge cushion on the blackboard of the film transfer clamp in sequence, and clamping the film transfer clamp. Note that PVDF membranes should be placed at protein locations of corresponding molecular weights and no bubbles should be created between each layer. Placing the clamps into a film transfer groove, placing ice bags at two sides of the film transfer groove, pouring film transfer liquid, setting current of 300mA, and setting time of 70min.

(7) Closing: blocking solutions were prepared with 1 XTBST and either cow milk or BSA. And after the film transfer is finished, taking out the PVDF film, and immersing the PVDF film in the sealing liquid as soon as possible to avoid drying the PVDF film. The mixture was blocked for 3h on a room temperature shaker.

(8) Incubating primary antibodies: in accordance with the stated proportions, primary antibodies were diluted with 1% skim milk or BSA, and each protein band incubated with 2mL antibody dilution at 4℃overnight.

(9) Incubating a secondary antibody: the strip was washed 3 times with 1 XTBST for 10min each. The secondary antibody was diluted with 1% skim milk or BSA at the ratio of the instructions, and each protein band was incubated with 2mL antibody dilution for 2h at 37 ℃.

(10) Developing: the strip was washed 3 times with 1 XTBST for 10min each. Each protein band was prepared with about 200. Mu.L of luminescence, developed with a Universal Hood II imager, and finally analyzed for gray scale values with quality 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 measured and the volume of 1. Mu.g RNA calculated. Bulge-Loop at 50. Mu.L of 20. Mu. Mol/L using reverse transcription kit of the Libo organism ^TM 150 mu L of RNase-free water is added into the miRNA RT Primer to prepare 5 mu mol/L of Bulge-Loop ^TM miRNA RT Primer. Experiments were performed in accordance with the system of table 5 (on ice):

Table 5:

after the above systems are mixed evenly, the mixture is centrifuged instantaneously, and 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 measured and the volume of 500ng RNA calculated. Experiments (on ice) were performed using the TaKaRa reverse transcription kit according to the table 6 system:

table 6:

* The reaction system can be scaled up as desired, and a maximum of 500ng Total RNA can be used in a 10. Mu.L reaction system.

After the above systems were mixed, the mixture was centrifuged instantaneously and the RT reaction procedure was 15min at 37℃for 5s at 85℃at 4 ℃.

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

Experiments were performed in accordance with the system of table 7 (on ice):

table 7:

qPCR reaction procedure is shown in table 8:

table 8:

primer sequences are shown in Table 9:

table 9:

gene	Upstream primer	Downstream primer
			miRNA-195-5p	5’-CCTAGCAGCACAGAA A-3’	5’-GAGCAGGCTGGAGAA-3’
U6	5’-CTCGCTTCGGCAGCACATA-3’	5’-CGCTTCACGAATTTGCGTG-3’
			mRNA-ERK	5’-TGAAGACACAGCACCTCAGCAATG-3’	5’-GGTGTTCAGCAGGTTGGAAG-3’
mRNA-GAPDH	5’-AAACCCATCACCATCTTCCA-3’	5’-GTGGTTCACACCCATCACAA-3’

10. Cell Al (mal) ₃ Fluorescence distribution detection: cells were spread on a laser co-polymerization Jiao Weikong dish, stained with aluminum when the cells were grown to 30%, and cultured 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 was fixed at room temperature for 20min with 1mL each dish. Then washed 3 times with 1% PBS for 5min each.

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

(4) 8-HQ stock solution was diluted 100-fold with DMSO to prepare 8-HQ working solution (500. Mu. Mol/L) for aluminum staining, 1mL each dish, and the dishes were placed in a cassette and incubated at 37℃for 30min. Then washed 3 times with 1% PBS for 5min each. The following steps should be performed in the absence of light.

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

(6) 1mL of 1% PBS was added to each dish and the assay was performed using laser confocal.

11. Tau protein immunofluorescence: cells were plated on a laser co-polymer Jiao Weikong cuvette and stained with aluminum or otherwise tampered with when the cells were 30% long.

(1) 10 (1) (2) (3) as above.

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

(3) 5% BSA was blotted, 1% BSA diluted primary antibody was added, 500. Mu.L per dish, and overnight at 4 ℃.

(4) The cells were washed 3 times with 1% PBS for 5min each. Fluorescent secondary antibodies diluted with 1% BSA were added and placed in a cassette and incubated at 37℃for 1h. The following steps should be performed in the absence of light.

(5) The cells were washed 3 times with 1% PBS for 5min each.

(6) And (5) and (6) of the same 10.

3. Statistical analysis: statistical analysis was performed using SPSS 22.0 software. All data are metering data, adopt

And (3) representing. The comparison among multiple groups adopts single-factor variance analysis; two-by-two comparison: the variance was verified using LSD and the variance was verified using Dunnett. Analysis of P-ERK and Al (mal) using factorial analysis ₃ Is described, is a single action and interactions of the same. Check level α=0.05 (double sided).

4. Results

1. Experimental results of different doses of aluminum maltol contamination

A. Distribution of aluminum maltol in PC12 cells after aluminum maltol treatment: detection of intracellular Al (mal) using laser confocal ₃ Is a distribution of (a). As shown in FIG. 1, the fluorescence signal shows that aluminum is mainly distributed in cytoplasm,and concentrate around the nucleus with Al (mal) ₃ The increase in concentration gradually increases the fluorescence signal.

B. Effect of aluminum maltol on PC12 cell viability and morphology: using Al (mal) of different concentrations ₃ Cells were treated (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) and after 24 hours cell viability was measured using CCK8 kit. The detection result is shown in fig. 2, and 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) ₃ Cell viability after exposure was 1, 92.05, 87.69, 85.62, 82.88, 78.67, 71.75, respectively. Wherein, al (mal) is contained in 100. Mu. Mol/L, 200. Mu. Mol/L, 400. Mu. Mol/L ₃ In the concentration treatment, the cell viability was maintained at 80% or more, so that 100. Mu. Mol/L, 200. Mu. Mol/L, 400. Mu. Mol/L of Al (mal) was selected ₃ The concentrations were used as low, medium and high dose groups.

Through 100 mu mol/L, 200 mu mol/L, 400 mu mol/LAl (mal) ₃ After treatment, the cell morphology was observed using a 100X inverted microscope, as shown in FIG. 3, and compared with the control group, the cell morphology was 100. Mu. Mol/LAl (mal) ₃ The cells of the group are slightly reduced but not obvious; 200 mu mol/LAl (mal) ₃ Reduced cell density, flattened cells, reduced third dimension, shorter axons, and reduced cell attachment; 400 mu mol/LAl (mal) ₃ The 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 increased.

C. Effect of aluminum maltol on PC12 cell miRNA-195-5p expression

The expression level of different groups of miRNA-195-5p is detected by adopting qT-PCR. The results are shown in Table 11 and FIG. 4, and the results show that the expression level of miRNA-195-5p decreases with the increase of the amount of the dye aluminum. Compared with the control group, 100 mu MAl (MAl) ₃ Group, 200. Mu.M Al (mal) ₃ Group, 400. Mu.M Al (mal) ₃ The groups were statistically different and decreased by 14.7%, 28.0%, 40.9% (P)<0.05). With 100 mu M Al (mal) ₃ Group contrast, 200. Mu.M Al (mal) ₃ And 400 mu M Al (mal) ₃ Group miRNA-195-5P expression level was significantly reduced (P)<0.05)。

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

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

D. Effect of aluminum maltol on PC12 cell mRNA-ERK, total ERK, P-ERK protein expression

The expression levels of different groups of mRNA-ERK were detected by qT-PCR. The results are shown in FIG. 5, which shows that with Al (mal) ₃ The concentration was increased, and the expression level of mRNA-ERK was not changed.

And detecting the relative expression quantity of the total ERK and the P-ERK proteins by using Western blot. The results are shown in Table 12, FIG. 5, FIG. 6, and show that the results follow Al (mal) ₃ The concentration is increased, the total ERK protein expression amount is unchanged, and the P-ERK protein expression amount is gradually increased. Compared with the control group, 100 mu M Al (mal) ₃ No significant differences in the expression levels of group P-ERK proteins occurred (P>0.05)；200μM Al(mal) ₃ The expression level of group P-ERK protein was increased 1.736 times (P<0.05)；400μM Al(mal) ₃ The expression level of the group P-ERK protein is increased by 1.993 times (P<0.05)。

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

Note that: the letter differences represent differences with statistical significance P <0.05.

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

And detecting the relative expression quantity of tau, PHF, NFT protein by using Western blot. The results are shown in FIG. 7, which shows that the tau, PHF, NFT expression level increases with the increase of the dyed aluminum concentration. Tau5 results showed 100. Mu.M Al (mal) compared to the control group ₃ No significant differences in the expression levels of histone tau protein occurred (P>0.05)，200μmol/LAl(mal) ₃ Group, 400. Mu. Mol/LAl (mal) ₃ The histone expression level was increased 1.220-fold and 1.459-fold (P)<0.05). PHF protein results showed 100. Mu. Mol/L Al (mal) compared to the control group ₃ Group, 200. Mu. Mol/L Al (mal) ₃ Group, 400. Mu. Mol/L Al (mal) ₃ The histone expression level was increased 1.184 times, 1.533 times, 2.500 times (P)<0.05). NFT protein results showed 100 μmal (MAl) compared to control ₃ No significant differences in group NFT protein expression levels occurred (P>0.05)，200μmol/L Al(mal) ₃ Group, 400. Mu. Mol/L Al (mal) ₃ The histone expression level was increased 1.394-fold and 2.417-fold (P)<0.05)。

F. Effect of aluminum maltol on tau5, PHF, NFT fluorescence distribution of PC12 cells

Immunofluorescence showed results as shown in FIGS. 8, 9, and 10, showing tau5 is localized mainly in the cytoplasm, PHF is localized mainly in the nucleus, NFT has distribution in both the nucleus and cytoplasm, and Al (mal) is followed ₃ Increasing the concentration of the fluorescent signalAnd gradually enhancing. Warp Al (mal) ₃ After treatment, tau5 and NFT both appear as larger fluorescent plaques in the cytoplasm; whereas PHF is a weak fluorescent signal in the cytoplasm of cells, where the transfer of protein from the nucleus to the cytoplasm occurs.

2. miRNA-195-5p intervention experimental result

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

Firstly, different miRNA-195-5p overexpression time groups are set, the qT-PCR result is shown in figure 11, and the result shows that the miRNA-195-5p overexpression has the highest expression level at 48 hours, so that the experiment is set to select miRNA-195-5p overexpression to intervene in the culture for 48 hours, and then the subsequent indexes are detected.

After miRNA-195-5p micrometers treatment, the cell morphology was observed with a 100X inverted microscope, and the results are shown in FIG. 12, which shows that Al (mal) was compared with the control group ₃ The cell number is gradually reduced by contamination, the axon is reduced and broken, the cell body is rounded, and after the overexpression treatment of miRNA-195-5p, the cell number is increased, the axis mutation is long, and the cell connection is increased.

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

The expression level of different groups of miRNA-195-5p is detected by adopting qT-PCR. The results are shown in Table 13 and FIG. 13, and the results show that compared with the control group, 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 increased by 1428.634 times (P)<0.05 A) is provided; with 200 mu mol/L Al (mal) ₃ In comparison to the group, miRNA-195-5p overexpressed +200. Mu. Mol/L Al (mal) ₃ The expression level of the group miRNA-195-5p is increased by 1826.305 times.

Table 13: expression level of miRNA-195-5p after miRNA-195-5p overexpression intervention

Group of	Expression level
		Control group	1.000±0.000 a
200μM Al(mal) 3	0.750±0.029 b
		Air white matter particle group	1.066±0.138 a
miRNA-195-5p overexpression group	1429.634±76.693 c
		miRNA-195-5p overexpression +200mu.M Al (mal) 3 Group of	1370.479±146.804 c

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

The expression levels of different groups of mRNA-ERK were detected by qT-PCR. The results are shown in Table 14 and FIG. 14, and the results show that the miRNA-195-5p overexpression does not cause the change of the mRNA-ERK expression level. NC mimcs group, 200. Mu.M Al (mal) compared to control group ₃ Group, miRNA-195-5p micrometers group, miRNA-195-5p micrometers+200. Mu. Mol/L Al (mal) ₃ No significant difference in the amount of mRNA-ERK expression occurred in the group.

Table 14: expression level of mRNA-ERK after miRNA-195-5p overexpression intervention

Note that: the letter differences represent differences with statistical significance P <0.05.

And detecting the total ERK and the P-ERK protein expression level by using a WesternBlot. The results are shown in FIG. 15The total ERK expression was shown to be unchanged from group to group; compared with the control group, the expression level of the blank particle group P-ERK is unchanged, 200 mu mol/L Al (mal) ₃ The expression level of the P-ERK protein in the affected group is increased by 1.736 times (P<0.05 The expression level of the miRNA-195-5P over-expression group P-ERK protein is reduced by 16.69 percent (P)<0.05 A) is provided; analysis of the factorial analysis shows that miRNA-195-5p is over expressed and Al (mal) ₃ Interaction with P-ERK protein expression, i.e., miRNA-195-5P is involved in Al (mal) ₃ Abnormal expression of the P-ERK protein.

D. Effect of miRNA-195-5p overexpression on PC12 cell tau, PHF, NFT protein expression

Western blot is adopted to detect relative expression quantity of tau5, PHF and NFT proteins. The results are shown in FIG. 16, which shows that the expression level of tau5, PHF and NFT proteins is unchanged, 200. Mu. Mol/L Al (mal) in the blank plasmid group compared with the control group ₃ The expression level of tau5, PHF and NFT proteins of the exposure group is respectively increased by 1.220 times, 1.533 times and 1.394 times (P)<0.05 miRNA-195-5P over-expression group tau5, PHF and NFT protein expression levels are respectively reduced by 13.900%, 21.310% and 18.270% (P)<0.05 A) is provided; analysis of the factorial analysis shows that miRNA-195-5p is over expressed and Al (mal) ₃ Interaction of tau5, PHF and NFT protein expression exists, namely miRNA-195-5p participates in Al (mal) ₃ The tau5, PHF and NFT proteins are abnormally expressed by contamination.

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

Immunofluorescence results are shown in FIGS. 17, 18, and 19, and show that tau5 is localized mainly in the cytoplasm, PHF is localized mainly in the nucleus, and NFT is distributed in both the nucleus and cytoplasm, both with Al (mal) ₃ The increasing concentration gradually increases the fluorescence signal. With 200 mu mol/L Al (mal) ₃ In comparison, tau5, PHF, NFT are in miRNA-195-5p micrometers+200. Mu. Mol/L Al (mal) ₃ The group fluorescent signal was reduced and the fluorescent plaque was reduced. Tau5 is mainly expressed as a decrease in overall fluorescence density after miRNA-195-5p micrometers are dried; PHF is mainly manifested by the disappearance of fluorescence signals in the cytoplasm; NFT is primarily manifested as massive fluorescent plaque dissipation in the cytoplasm.

The PC12 cells used in the invention are a kind of swelling of the sympathetic nervous systemTumor cells. The method has good and stable growth performance, and is suitable for gene transfection research. Furthermore, the nerve growth factor (nerve growth factor, NGF) receptor exists on the cell membrane of PC12, and can recognize the growth factor (NGF), grow neurites, and differentiate into cells with sympathetic neuron characteristics. Therefore, the present study uses the cells for molding, and Al (mal) ₃ Cell contamination was performed and entry of aluminum into the cell was detected by immunofluorescence and was distributed mainly in the cytoplasm and axons.

Effect of Al on tau protein hyperphosphorylation: aluminum is ubiquitous in life, but is not essential to life, and participates in biochemical reactions of the organism as a recognized neurotoxic metal, and aluminum can pass through blood brain barrier and placenta barrier to accumulate in the hippocampus, frontal cortex and other areas of brain, and repeatedly show accumulation in cognitive impairment susceptible neuron foci, and X-ray spectrum demonstrates accumulation of aluminum in neurofibrillary tangles formed in the brain of cognitive dysfunction patients. Furthermore, researchers have performed metal distribution investigation on brain tissues of patients diagnosed with AD, and the results indicate that there is aluminum deposition in the brain tissues. This all demonstrates that aluminum plays an important role in cognitive decline.

According to the invention, an aluminum-dyed cell model is constructed, whether aluminum can enter cells or not is detected through 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-dyed cells, so that in-vitro research can be verified, the aluminum-dyed cells can be built into the model of accumulation of the aluminum in neurons, and the change of related molecules can be detected.

The invention adopts tau5 antibody, tau-pSer396 antibody and AT8 (tau-pSer 202, thr 205) antibody to detect total tau, PHF and NFT respectively, so as to investigate the expression condition of tau AT different phosphorylation sites. The results show that tau5 protein expression increases with increasing aluminum concentration, PHF at low doses (100. Mu. Mol/LAl (mal) ₃ ) A significant increase was seen when the NFT was only up to a medium dose (200. Mu. Mol/L Al (mal)) ₃ ) A significant increase was observed only when this was the case, so we speculate that although aluminium may cause hyperphosphorylation of tau protein, PHF is more sensitive to aluminium toxicity, early in timeI.e., may occur while NFT occurs later. Furthermore immunofluorescence showed that total Tau represented by Tau5 antibody was detected mainly in the cytoplasm, and that total Tau signal was enhanced after staining with aluminium, while its localization was unchanged. Antibodies directed against phosphorylated tau show localization of tau in the nucleus. PHF is mainly located in the nucleus, but it appears in the cytoplasm after aluminum staining, and nuclear signaling is also enhanced. NFT is distributed in both the nucleus and cytoplasm, but after aluminum staining, is predominantly manifested by an increase in fluorescence signal in the cytoplasm. Under the light microscope, the cell axons are obviously shortened after the aluminum is dyed, the cell connection is reduced, and the cells are obviously reduced.

The above results all demonstrate that aluminium can cause abnormal phosphorylation of tau protein, leading to abnormal aggregation of tau protein, leading to cytotoxicity. Thus, we propose that aluminium can cause tau protein hyperphosphorylation, and that aluminium phosphorylates tau protein mainly first occurs at the Ser396 site, with increasing aluminium concentration at Ser202 and Thr205 sites.

Inhibition of ERK activation effect on Al-induced protein hyperphosphorylation: extracellular signal-regulated kinases (Extracel lular s ignal regulated kinase, ERK) belong to the family of mitogen-activated protein kinases (mi togen act ivated prote in kinase, MAPK), play a role in the signaling cascade, transmitting extracellular signals to intracellular targets.

Extracellular signal-regulating kinases ERK are largely divided into two types, ERK1 and ERK2, which are evolutionarily conserved, ubiquitous serine-threonine kinases that regulate cellular signals under normal and pathological conditions. The phosphorylation sites of Tau proteins are predominantly threonine (pThr) or serine (pSer) sites, so we infer that aluminium-induced hyperphosphorylation of Tau proteins may be effected by activation of ERK.

In the present invention, al (mal) is first performed on cells ₃ Contamination found that 200. Mu.M Al (mal) compared to the control group ₃ Group sum 400. Mu.M Al (mal) ₃ The expression level of the group P-ERK protein is increased by 1.736 times and 1.993 times respectively (P<0.05). Furthermore, tau5 protein, PHF and NFT were expressed in 200. Mu.M Al (mal) ₃ Significant increases were seen in both groups. The results indicate that Al (mal) ₃ The contamination can cause ERK to be effectiveThe effect of pathway hyperphosphorylation is consistent with abnormal tau phosphorylation suggesting that we have the possibility that hyperphosphorylation of the ERK pathway is an upstream signal leading to tau hyperphosphorylation and is one of the reasons for aluminum-induced tau hyperphosphorylation and cytotoxicity of PC12 cells.

Conclusion: aluminum maltol can enter cells and is mainly distributed in cytoplasm; tau5 protein is predominantly distributed in the cytoplasm; double helix filaments (PHF, p-Ser 396) are mainly distributed in the nucleus; neurofibrillary tangles (NFT, p-Ser202, p-Thr 205) are distributed in both the nucleus and cytoplasm. And tau5 and NFT both show increased protein expression in cytoplasm after infection of aluminum maltol, wherein PHF not only has increased expression quantity but also has the phenomenon of transferring to cytoplasm.

The phosphorylation of tau protein by aluminum occurs primarily at the Ser396 site, with the phosphorylation at the Ser202, thr205 site occurring as the aluminum concentration increases.

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

Action of miRNA-195-5p to regulate aluminium-induced tau protein hyperphosphorylation:

the invention adopts PC12 cells to screen miRNA of cells of an aluminum-dyed group and cells of a control group, and discovers that miRNA-195-5p is down-regulated in the expression of the aluminum-dyed group and the difference has statistical significance. In addition, by establishing low, medium and high-dyeing aluminum cell models, the expression quantity of miRNA-195-5p is found to be gradually reduced along with the increase of the concentration of the dyed aluminum, and the difference is statistically significant. The results indicate that aluminum in PC12 cells can affect the expression of miRNA-195-5 p.

We speculate that miRNA-195 may improve cognitive impairment by inhibiting protein kinase activation, reducing aluminum-induced tau hyperphosphorylation. 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 level of total phosphorylated tau protein, PHF and NFT in cells transfected with miRNA-195-5p mimics is reduced, and the results indicate that the phosphorylation of tau protein depends on the expression of miRNA-195-5p,expression of miRNA-195-5p was suggested to be inversely related to the extent of tau protein phosphorylation. Simultaneous miRNA-195-5p overexpression and 200. Mu. Mol/L Al (mal) ₃ Treatment, analysis of factorial showed miRNA-195-5p overexpression and 200. Mu. Mol/L Al (mal) ₃ Interaction with tau5, PHF, NFT expression indicates that miRNA-195-5p is involved in Al (mal) ₃ Resulting abnormal phosphorylation of tau protein.

After miRNA-195-5P is over expressed, mRNA-ERK and total ERK proteins in cells are not changed, and only the expression level of P-ERK is reduced, so that miRNA-195-5P can influence 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 fluorescent plaques are reduced; under the light microscope, the cell damage is reduced, the axis mutation is long, and the cell contact is increased, which indicates that the cell damage caused by aluminum can be saved by the overexpression of miRNA-195-5 p. In conclusion, aluminium-induced tau hyperphosphorylation in PC12 cells may be exerted by inhibiting miRNA-195 expression, resulting in increased ERK activation.

To sum up, al (mal) ₃ Contamination can inhibit the expression of PC12 cell miRNA-195-5p, leading to aberrant activation of ERK, leading to tau protein hyperphosphorylation.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Sequence listing

<110> university of Shanxi medical science

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

<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 (4)

1. The application of miRNA-195-5p in preparing an agent for inhibiting or reducing nerve cell damage caused by aluminum maltol is characterized in that: the miRNA-195-5p overexpression inhibits ERK activation, reduces tau protein phosphorylation, inhibits or reduces nerve cell damage caused by aluminum maltol, reduces nerve cell damage, and increases axis mutation and cell contact;

   the tau protein is abnormally phosphorylated to occur at the Ser396 site and at the Ser202 and Thr205 sites.
2. 2. The use of miRNA-195-5p according to claim 1 for the preparation of an agent for inhibiting or reducing neurocyte damage caused by aluminum maltol, characterized in that: and the miRNA-195-5p is over-expressed for 36 hours to inhibit or reduce nerve cell damage caused by aluminum maltol.
3. 3. The use of miRNA-195-5p according to claim 1 for the preparation of an agent for inhibiting or reducing neurocyte damage caused by aluminum maltol, characterized in that: the nucleotide sequence of the miRNA-195-5p is shown as SEQ ID NO. 1.
4. 4. The use of miRNA-195-5p according to claim 1 for the preparation of an agent for inhibiting or reducing neurocyte damage caused by aluminum maltol, characterized in that: the upstream primer of the miRNA-195-5p is as follows: 5'-CCTAGCAGCACAGAA A-3'; the downstream primer is: 5'-GAGCAGGCTGGAGAA-3'.

Images