Application of melatonin in preparation of medicine for treating learning and memory disorder diseases caused by nerve cell injury or mitochondrial fission

Abstract

The invention relates to the field of pharmacy, in particular to application of melatonin to preparation of a medicine for treating learning and memory disorder diseases caused by nerve cell injury or mitochondrial fission. Experiments prove that the melatonin can inhibit mitochondrial fission, increase ATP generation and mitochondrial membrane potential level, down-regulate ROS level, improve cell viability, improve mitochondrial dysfunction caused by fluorine-aluminum combined exposure and promote synaptic protein GAP-43 and Ng expression by promoting SIRT1 / PGC-1alpha pathway, so that the melatonin can inhibit mitochondrial fission, increase ATP generation and mitochondrial membrane potential level, reduce ROS level, improve cell viability, improve mitochondrial dysfunction caused by fluorine-aluminum The melatonin can be used for preparing medicines for treating or improving learning and memory disorder diseases caused by nerve cell injury or mitochondrial fission caused by fluorine and aluminum combination.

Description

Technical Field

[0001] The invention relates to the field of pharmacy, and in particular to the application of melatonin in the preparation of medicines for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission. Background Art

[0002] Fluorine (F) is a non-metallic element widely found in nature. It is chemically active and often exists in the form of compounds. Studies have shown that excessive fluoride can damage brain structure and learning and memory, but its mechanism is not yet fully understood. Aluminum (Al) is one of the most widely distributed metals in the environment. Excessive aluminum can cause changes in rats' neurobehavioral abilities and damage their learning and memory abilities.

[0003] Fluoride and aluminum are both harmful to the body and have similar target organs. Compared with single fluoride or aluminum exposure, the impact of co-exposure to fluoride and aluminum in the environment on human health is equally worthy of attention. Fluoride and aluminum can form complexes after ingestion, which are more likely to cross the blood-brain barrier and be stored in the brain, impairing learning and memory abilities. Epidemiological studies have shown that high levels of fluoride and aluminum intake in drinking water are associated with the risk of dementia. Fluoride-aluminum complexes accumulate in the brain and induce mitochondrial fission, impair the activity of enzymes related to energy production, disrupt the balance of free radicals, lead to lipid peroxidation and abnormal DNA repair mechanisms, and cause neurotoxicity, synaptic damage, and neurodegeneration. Neurotoxicity caused by combined exposure to fluoride and aluminum seriously affects the health of the exposed population, causing damage to nerve cells and a variety of neurological diseases. Therefore, it is necessary to develop disease-related pathways for the treatment of learning and memory disorders caused by combined exposure to fluoride and aluminum, which can cause nerve cell damage or mitochondrial fission. Summary of the invention

[0004] In view of the technical problems existing in the above-mentioned prior art, the purpose of the present invention is to provide the use of melatonin in the preparation of a drug for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission. The present invention verifies through experiments that melatonin can improve synaptic damage and nerve cell damage induced by fluorine-aluminum combined exposure, reduce oxidative stress of nerve cells induced by fluorine-aluminum combined exposure, improve cellular energy metabolism disorders, reverse mitochondrial fission of nerve cells induced by fluorine-aluminum combined exposure by upregulating the SIRT1 / PGC-1α pathway, and exert a neuroprotective effect, thereby improving learning and memory disorders caused by fluorine-aluminum combined exposure.

[0005] The present invention provides an application of melatonin in preparing a medicine for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission.

[0006] Furthermore, the melatonin is used to prepare a drug for treating nerve cell damage caused by combined exposure to fluorine and aluminum or learning and memory disorders caused by mitochondrial fission.

[0007] Furthermore, the drug contains melatonin as the only active ingredient.

[0008] Furthermore, the drug also includes pharmaceutically acceptable excipients and / or carriers.

[0009] Furthermore, the dosage form of the drug includes tablets, powders, granules, capsules or injections.

[0010] Furthermore, the injection is a solution or an emulsion.

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

[0012] Furthermore, the melatonin is used to prepare an expression promoter of synaptic protein GAP-43 or Ng.

[0013] Furthermore, the expression promoter of the synaptic protein GAP-43 or Ng is used to prepare a drug for improving nerve cell damage.

[0014] In summary, compared with the prior art, the present invention has the following advantages and effects: The present invention discloses for the first time that melatonin can be used to prepare a drug for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission due to combined fluorine and aluminum exposure. Melatonin promotes the SIRT1 / PGC-1α pathway, inhibits mitochondrial fission, improves mitochondrial dysfunction caused by combined fluorine and aluminum exposure, promotes GAP-43 and Ng expression, and further improves nerve cell damage caused by combined fluorine and aluminum exposure and learning and memory disorders caused by mitochondrial fission. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Effects of combined exposure to fluorine and aluminum on the level of melatonin (Mel) in NG108-15 cells; a indicates comparison with the control group, p <0.05; b means compared with Al group, p <0.05.

[0016] Figure 2 The effect of different concentrations of melatonin on the viability of NG108-15 cells, a represents the comparison with the control group, p <0.05.

[0017] Figure 3 The effect of melatonin on the viability of NG108-15 cells exposed to fluorine and aluminum; a means compared with the control group, p <0.05; b means compared with the FA (fluorine + aluminum) group, p <0.05.

[0018] Figure 4 The effect of melatonin on the expression of GAP-43 and Ng in NG108-15 cells exposed to fluorine and aluminum; Figure A is the relative expression of GAP-43 mRNA; Figure B is the relative expression of Ng mRNA; Figure C is the band diagram of GAP-43 and Ng proteins; Figure D is the statistical diagram of the relative expression of GAP-43 protein; Figure E is the statistical diagram of the relative expression of Ng protein; a means compared with the control group, p <0.05; b means compared with the FA group, p <0.05.

[0019] Figure 5 The effect of melatonin on the level of reactive oxygen species (ROS) in NG108-15 cells exposed to fluorine and aluminum; Figure A is a representative diagram of ROS in NG108-15 cells detected by flow cytometry; Figure B is the relative expression level of ROS; a means compared with the control group, p <0.05; b means compared with the FA group, p <0.05.

[0020] Figure 6 The effect of melatonin on ATP level in NG108-15 cells exposed to fluorine and aluminum; a means compared with the control group, p <0.05; b means compared with the FA group, p <0.05.

[0021] Figure 7 The figure shows the effect of melatonin on the mitochondrial membrane potential of NG108-15 cells exposed to fluoride and aluminum. JC-1aggregates corresponds to the representative fluorescence image of JC-1 polymer, JC-1 monomer corresponds to the representative fluorescence image of JC-1 monomer, and Merge corresponds to the representative fluorescence image of JC-1 polymer and JC-1 monomer.

[0022] Figure 8 Figure 2 shows the effect of melatonin on the expression of Drp1, Fis1 and MFF in NG108-15 cells exposed to fluorine and aluminum. Figure A shows the relative expression of Drp1 mRNA. Figure B shows the relative expression of Fis1 mRNA. Figure C shows the protein bands of Drp1, Fis1 and MFF. Figure D shows the statistical graph of the relative expression of Drp1 protein. Figure E shows the statistical graph of the relative expression of Fis1 protein. Figure F shows the statistical graph of the relative expression of MFF protein. a means compared with the control group, p <0.05; b means compared with the FA group, p <0.05.

[0023] Fig. 9Figure 2 is the effect of melatonin on the expression of SIRT1 and PGC-1α in NG108-15 cells exposed to fluorine and aluminum; Figure A is the relative expression of SIRT1 mRNA; Figure B is the relative expression of PGC-1α mRNA; Figure C is the protein band diagram of SIRT1 and PGC-1α; Figure D is the statistical diagram of the relative expression of SIRT1 protein; Figure E is the statistical diagram of the relative expression of PGC-1α protein; a means compared with the control group, p <0.05; b means compared with the FA group, p <0.05.

[0024] Fig.10 The effect of melatonin and PGC-1α siRNA on the expression of PGC-1α and Drp1 in NG108-15 cells exposed to fluorine and aluminum; Figure A is the relative expression of PGC-1α mRNA; Figure B is the relative expression of Drp1 mRNA; Figure C is the PGC-1α and Drp1 protein band diagram; Figure D is the relative expression of PGC-1α protein; Figure E is the relative expression of Drp1 protein. A indicates that compared with the (FA + Control siRNA) group, p <0.05; b indicates comparison with (FA + Mel + Control siRNA) group, p <0.05; c indicates comparison with (FA + PGC-1α siRNA) group, p <0.05.

[0025] Fig.11 Figure 2 is the effect of melatonin and PGC-1α siRNA on ROS in NG108-15 cells exposed to fluorine and aluminum; Figure A is a representative diagram of ROS in NG108-15 cells detected by flow cytometry; Figure B is the relative expression level of ROS in NG108-15 cells; a means compared with the (FA + Control siRNA) group, p <0.05; b indicates comparison with (FA + Mel + Control siRNA) group, p <0.05; c indicates comparison with (FA + PGC-1α siRNA) group, p <0.05.

[0026] Fig.12 The effect of melatonin and SIRT1 siRNA on the expression of PGC-1α in NG108-15 cells exposed to fluorine and aluminum; Figure A is the representative band diagram of SIRT1 and PGC-1α proteins; Figure B is the relative expression of PGC-1α protein; Figure C is the relative expression of PGC-1α protein. A indicates that compared with the (FA + Control siRNA) group, p<0.05; b indicates comparison with (FA + Mel +Control siRNA) group, p <0.05; c indicates comparison with (FA + SIRT1 siRNA) group, p <0.05. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0028] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0029] Fluoride and aluminum are both harmful to the body and have similar target organs. Compared with single fluoride or aluminum exposure, the impact of co-exposure to fluoride and aluminum in the environment on human health is equally worthy of attention. After ingestion, fluoride and aluminum can form a complex, which is easier to cross the blood-brain barrier and be stored in the brain, impairing learning and memory ability. The accumulation of aluminum fluoride complexes in the brain induces mitochondrial fission, impairs the activity of enzymes related to energy production, destroys the balance of free radicals, leads to lipid peroxidation and abnormal DNA repair mechanisms, and causes neurotoxicity, synaptic damage and neurodegeneration. Neurotoxicity caused by combined exposure to fluoride and aluminum seriously affects the health of the exposed population, causing nerve cell damage and a variety of neurological diseases. Therefore, it is necessary to develop disease-related pathways for the treatment of learning and memory disorders caused by combined exposure to fluoride and aluminum, which causes nerve cell damage or mitochondrial fission.

[0030] The present invention provides an application of melatonin (Mel) in the preparation of a drug for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission. The inventor's research team found in previous studies that combined exposure to fluorine and aluminum inhibits the SIRT1 / PGC-1α pathway, promotes mitochondrial fission, and leads to mitochondrial dysfunction and nerve cell damage, which manifests as learning and memory disorders. The present invention first determined the content of Mel in cells exposed to fluorine and aluminum together, analyzed the effect of fluorine and aluminum together exposure on the content of Mel in nerve cells, and then detected the effect of Mel intervention on nerve cell vitality, the expression levels of GAP-43, Ng, nerve cell ROS, ATP, mitochondrial membrane potential, Drp1, Fis1, MFF and SIRT1 / PGC-1α pathway. It was found that Mel promoted the SIRT1 / PGC-1α pathway, inhibited mitochondrial fission, increased ATP generation and mitochondrial membrane potential levels, downregulated ROS levels, improved cell vitality, improved mitochondrial dysfunction caused by fluorine and aluminum together exposure, promoted the expression of synaptic proteins GAP-43 and Ng, and had a protective effect on nerve cell damage caused by fluorine and aluminum together exposure, thereby improving the learning and memory disorders caused by fluorine and aluminum together exposure.

[0031] Example 1: Use of melatonin in the preparation of a drug for treating nerve cell damage caused by combined exposure to fluoride and aluminum 1. Experimental Methods 1. Cell experiment grouping and processing (1) Establishment of fluoride-aluminum combined exposure cell model and grouping Select NaF 1 / 2 IC 50 (40 mg / L) and 1 / 2 IC of AlCl3 50 (160 mg / L) was used as the contamination concentration. The specific experimental groups and contamination concentrations are shown in Table 1.

[0032] Table 1 List of cell experiment groups (2) Determination of Mel concentration 1) Mel solution preparation: Dissolve Mel in DMSO to prepare a stock solution with a concentration of 10 mM / L, and store it in a -20℃ refrigerator away from light. Before each experiment, dilute the Mel stock solution with culture medium.

[0033] 2) Determination of the effect of Mel on NG108-15 cell viability: Mouse neuroblastoma cells (NG108-15) in logarithmic growth phase and normal cell status were taken and their density was adjusted to 1.0×10 5 / mL, 100 μL / well was inoculated in a 96-well plate. When the cells adhered to the wall and grew to 80%, 100 μL of DMEM medium containing Mel concentrations of 12.5 μmol / L, 25 μmol / L, 50 μmol / L, 75 μmol / L, 100 μmol / L, 200 μmol / L, 300 μmol / L, and 400 μmol / L was replaced. Each group had 6 parallel wells. After culturing in a 37°C incubator for 24 hours, 10 μL of CCK-8 reagent was added to each well, and the cells were placed in a 37°C incubator for incubation for 1 hour. The OD value was measured at a wavelength of 450 nm using an enzyme reader to calculate the viability of NG108-15 cells.

[0034] (3) Experimental grouping of NG108-15 cell model exposed to Mel and fluoride-aluminum A Mel concentration of 100 μmol / L was selected for intervention. The specific experimental groups are shown in Table 2.

[0035] Table 2 List of groups in the Mel intervention experiment (4) Treatment of NG108-15 cell model with combined exposure to Mel and fluoride-aluminum After the subcultured NG108-15 cells were placed in an incubator for 24 hours, the culture medium was removed and replaced with DMEM culture medium containing 100 μmol / L Mel according to the grouping in Table 1 or Table 2, and the culture was continued for 4 hours. Then, according to the corresponding poisoning dose and grouping, the DMEM culture medium containing NaF and AlCl3 was replaced; or the DMEM culture medium containing NaF, AlCl3, and Mel was used to extract the total RNA or protein of NG108-15 cells after continuing to culture for 24 hours; the viability, ROS, ATP, and mitochondrial membrane potential of NG108-15 cells were detected.

[0036] (5) Cell transfection experiment grouping and treatment Select 1 / 2 IC of NaF and AlCl3 50 As the poisoning concentration, Mel 100 μmol / L was used as the intervention concentration of NG108-15 cells. The specific experimental groups are shown in Table 3.

[0037] After cell transfection, the cells were replaced with DMEM medium containing NaF and AlCl3 according to the determined poisoning dose and grouping, and used for RNA, protein, and ROS detection 24 hours after poisoning; or replaced with DMEM medium containing 100 μmol / L Mel for 4 hours and then replaced with DMEM medium containing Mel, NaF, and AlCl3, and then cultured for another 24 hours for RNA, protein, and ROS detection.

[0038] Table 3 List of cell transfection experiment groups Note: + indicates treatment has been carried out, - indicates no treatment has been carried out.

[0039] 2. Indicators and their detection methods (1) ELISA to detect the Mel level in the supernatant of NG108-15 cell culture medium 1) Follow the instructions of the mouse melatonin ELISA kit (purchased from Shanghai Fankewei Biotechnology Co., Ltd.) and remove the strips from the aluminum foil bag after equilibration at room temperature for 20 minutes.

[0040] 2) Add 50 μL of standards of different concentrations to the standard wells.

[0041] 3) Add 10 μL of the sample to be tested and 40 μL of sample diluent to each sample well (50 μL in total per well).

[0042] 4) Add horseradish peroxidase (HRP)-labeled antibodies to each standard well and sample well, 100 μL per well, carefully seal each reaction well with a sealing film, protect from light, and incubate at 37°C for 1 hour.

[0043] 5) Add PBS to the plate to wash, discard the PBS, and gently pat dry on absorbent paper. Wash the plate 5 times in total.

[0044] 6) Add 50 μL of substrate A and 50 μL of substrate B to each well, protect from light, and incubate at 37 °C for 15 min.

[0045] 7) After incubation, add 50 μL of stop solution to each well and measure the OD value at 450 nm using a microplate reader. Calculate the sample content using the standard curve equation.

[0046] (2) qRT-PCR mRNA expression levels of GAP-43, Ng, Drp1, Fis1, SIRT1, and PGC-1α in NG108-15 cells Three replicate wells were set up for each sample, and the experiment was repeated at least 3 times.

[0047] 1) Primer design and synthesis The sequences of SIRT1, PGC-1α, Drp1, Fis1, GAP-43, Ng, and GAPDH primers are shown in Table 4 .

[0048] Table 4 qRT-PCR primer sequence list 2) The qRT-PCR reaction system is shown in Table 5.

[0049] Table 5 qRT-PCR reaction system 3) qRT-PCR reaction conditions are shown in Table 6.

[0050] Table 6 qRT-PCR reaction conditions Note: - indicates that the corresponding step will not be cycled.

[0051] 4) Calculation result: 2 -ΔΔCt The relative expression level of mRNA was calculated by the method. Among them, ΔΔCt = (Ct value of target gene in the test group - Ct value of internal reference gene in the test group) - (Ct value of target gene in the control group - Ct value of internal reference gene in the control group).

[0052] (3) Western Blot detection of protein expression of GAP-43, Ng, Drp1, Fis1, MFF, SIRT1, and PGC-1α in NG108-15 cells 1) Install the clean glass plate on the glue rack.

[0053] 2) Pour the prepared stacking gel and separation gel into the glass plate respectively, insert the comb, and after the stacking gel is completely agglutinated, install the glass plate vertically in the electrophoresis tank, pour the electrophoresis buffer (1×) into the electrophoresis tank, and remove the comb.

[0054] 3) SDS-PAGE electrophoresis: Add an equal amount of protein sample (10 μL) to each well, and add 2~3 μL of protein marker. Run the concentrated gel at 80 V (25 min), and adjust the voltage to 120 V for separation gel. Stop the electrophoresis when the sample reaches the bottom. Rinse the glass plate with pure water, remove the separation gel, and cut off the concentrated gel.

[0055] 4) Transfer: Soak the polyvinylidene fluoride (PVDF) membrane in methanol for 15 s and pure water for 10 min, attach it to the separation gel, and transfer it at 300 mA for 65 min.

[0056] 5) Blocking: After the transfer is completed, place the PVDF membrane into QuickBlock TM After completion, place the tube in the Western blocking solution and shake gently for 60 minutes.

[0057] 6) Primary antibody incubation: After blocking, wash the PVDF membrane three times with TBST and then incubate in the corresponding primary antibody at 4°C overnight. The internal reference is β-actin. The primary antibody dilution concentrations are: GAP-43 (1:1000); Ng (1:1000); Drp1 (1:1000); p-Drp1 (Ser637) (1:1000); Fis1 (1:1000); MFF (1:1000); SIRT1 (1:1000); PGC-1α (1:1000); β-actin (1:10000).

[0058] 7) Secondary antibody incubation: After primary antibody incubation, wash with TBST, wash on a shaker for 5 min each time, for a total of 3 times. After absorbing the water on the PVDF membrane with filter paper, put it in the corresponding secondary antibody, incubate the secondary antibody (1:10000) at room temperature, and incubate on a shaker for 1 hour. Wash the PVDF membrane after incubation with TBST 5 times, 5 min / time.

[0059] 8) Luminescent development: After immersing the PVDF membrane in the developer, place it in the gel imaging system for exposure. After image acquisition, use Image-J to analyze the image and calculate the grayscale value (the protein expression level is expressed by the ratio of the grayscale value of non-phosphorylated protein / the grayscale value of β-actin and the ratio of the grayscale value of phosphorylated protein / the grayscale value of non-phosphorylated protein).

[0060] (4) Detection of ROS levels in NG108-15 cells by flow cytometry 1) After the infection, remove the culture medium and add an appropriate amount of 0.25% trypsin to the 6-well plate to digest the cells. After the digestion is complete, add DMEM culture medium (containing 10% fetal bovine serum) to terminate the digestion reaction.

[0061] 2) Carefully transfer the resuspended cells to a new centrifuge tube, centrifuge (4°C, 300 rpm, 5 min), and slowly pour off the supernatant.

[0062] 3) Preheat PBS at 37°C, wash the cells twice by gently shaking, centrifuge (4°C, 300 rpm, 5 min), carefully discard the supernatant, and collect the cells after centrifugation.

[0063] 4) Dilute DCFH-DA at 1:1000, add 1 mL of serum-free DMEM medium (pre-loaded with 10 μmol / L DCFH-DA probe) to each well, resuspend the cells, and adjust the final cell concentration to 1×10 6 Pieces / mL.

[0064] 5) Incubate in dark place at 37°C for 20 min.

[0065] 6) Wash the cells after incubation with serum-free DMEM medium for 3 times. Detect ROS levels using flow cytometry.

[0066] (5) Chemiluminescence detection of ATP levels in NG108-15 cells of each group 1) After the infection, remove the culture medium and add lysis buffer to fully lyse the cells.

[0067] 2) Centrifuge at 4°C and 12,000 rpm for 5 min, then carefully draw the supernatant into a new EP tube.

[0068] 3) Dilute the ATP detection reagent to prepare the ATP detection working solution. Add the ATP standard solution to the ATP detection lysate on ice and dilute the solution in a gradient according to the instructions to draw a standard curve.

[0069] 4) Add 100 μL of ATP assay working solution to each well of the 96-well plate and incubate at room temperature for 5 min.

[0070] 5) Add the standard or sample (20 μL / well) and mix thoroughly to avoid bubbles. Use an ELISA reader to detect the chemiluminescence value in each well to be measured.

[0071] (6) Observation of mitochondrial membrane potential of NG108-15 cells in each group under fluorescence microscopy The mitochondrial membrane potential detection kit (purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.) was used as described in the instructions. Finally, the mitochondrial membrane potential of NG108-15 cells was observed using a fluorescence microscope and images were collected for analysis.

[0072] 2. Experimental Results 1. Effects of combined fluoride and aluminum exposure on Mel levels in NG108-15 cells In order to evaluate the effect of combined exposure to fluorine and aluminum on the Mel content in NG108-15 cells, Elisa was used to detect the concentration of Mel in the supernatant of NG108-15 cell culture medium. The results are shown in Figure 2. Figure 1 shown.

[0073] from Figure 1 It can be seen that compared with the control group, the concentration of Mel in the supernatant of NG108-15 cell culture medium in each experimental group was reduced, and the difference was statistically significant ( p <0.05); compared with group F, the concentration of Mel in the supernatant of NG108-15 cell culture medium in groups Al and FA increased, and the difference was statistically significant ( p <0.05); there was no significant change between the Al group and the FA group.

[0074] The results showed that combined exposure to fluorine and aluminum reduced the Mel concentration in NG108-15 cells.

[0075] 2. Exogenous supplementation of Mel reverses NG108-15 cell damage caused by combined exposure to fluoride and aluminum (1) Exogenous supplementation of Mel promotes the survival of NG108-15 cells exposed to fluoride and aluminum Based on the results that fluoride-aluminum combined exposure reduced the concentration of Mel in NG108-15 cells, the CCK-8 method was used to detect the effect of Mel on cell viability. The results are as follows Figure 2 shown.

[0076] from Figure 2 It can be seen that compared with the control group, the viability of NG108-15 cells in the 200μmol / L, 300μmol / L, and 400μmol / L Mel-treated groups decreased, and the difference was statistically significant ( p <0.05); the cell viability of the 100 μmol / L Mel-treated group was 97.937%, which was not significantly different from that of the control group. 100 μmol / L Mel was selected as the intervention concentration for subsequent in vitro experiments.

[0077] In order to evaluate the improvement effect of Mel intervention on the survival rate of NG108-15 cells exposed to fluorine and aluminum, Mel was used to intervene in the NG108-15 cells in the FA group and the cell viability was detected. The results are shown in Figure 3 shown.

[0078] from Figure 3 It can be seen that compared with the control group, the viability of NG108-15 cells in the FA group and FA + Mel group was reduced, and the difference was statistically significant ( p <0.05); compared with the FA group, the viability of NG108-15 cells in the FA + Mel group increased, and the difference was statistically significant ( p <0.05).

[0079] The results showed that Mel could alleviate the decreased viability of NG108-15 cells induced by combined exposure to fluorine and aluminum.

[0080] (2) Exogenous supplementation of Mel promotes the expression of synaptic proteins in NG108-15 cells exposed to fluorine and aluminum Nerve cells are interconnected through synapses. Synaptic damage is the basis of neurodegenerative diseases, and synaptic loss is associated with cognitive decline. Synaptic proteins Growth-associated protein-43 (GAP-43) and neurogranin (Ng) are two proteins involved in synaptic plasticity and learning and memory functions. GAP-43 expression is reduced in the brains of patients with dementia and in the hippocampus of mice with cognitive impairment caused by a low-fiber diet. Ng has been used as a biomarker for studying AD and aging. In order to further evaluate the effect of Mel on the expression of synaptic proteins in NG108-15 cells exposed to fluorine and aluminum, the mRNA expression levels of GAP-43 and Ng were detected after exogenous supplementation of Mel.

[0081] The results are as follows Figure 4 As shown in Figures A and B, compared with the control group, the mRNA of GAP-43 and Ng in NG108-15 cells in the FA group were downregulated, and the difference was statistically significant ( p <0.05); compared with the FA group, the mRNA expressions of GAP-43 and Ng in NG108-15 cells in the FA + Mel group were upregulated, and the difference was statistically significant ( p <0.05).

[0082] The results showed that Mel could alleviate the inhibitory effect of combined fluoride and aluminum exposure on the mRNA expression of GAP-43 and Ng in NG108-15 cells.

[0083] GAP-43 and Ng proteins are the executors of synaptic function. The present invention further evaluates the expression levels of GAP-43 and Ng at the protein level.

[0084] The results are as follows Figure 4As shown in Figures C, D, and E, compared with the control group, the proteins of GAP-43 and Ng in NG108-15 cells in the FA group were downregulated, and the difference was statistically significant ( p <0.05); compared with the FA group, the protein expressions of GAP-43 and Ng in NG108-15 cells in the FA + Mel group were upregulated, and the difference was statistically significant ( p <0.05).

[0085] The results showed that Mel could alleviate the inhibitory effect of combined fluoride and aluminum exposure on the expression of GAP-43 and Ng proteins in NG108-15 cells, and Mel could improve the synaptic damage induced by combined fluoride and aluminum exposure.

[0086] 3. Exogenous supplementation of Mel improves mitochondrial function of NG108-15 cells exposed to fluoride and aluminum (1) Exogenous supplementation of Mel down-regulates ROS levels in NG108-15 cells exposed to fluoride and aluminum To evaluate the effect of Mel on ROS generation in NG108-15 cells, the ROS levels were detected.

[0087] The results are as follows Figure 5 As shown, compared with the control group, the ROS generation of NG108-15 cells in the FA group and FA + Mel group increased, and the difference was statistically significant ( p <0.05); compared with the FA group, the ROS generation of NG108-15 cells in the FA + Mel group was reduced, and the difference was statistically significant ( p <0.05).

[0088] The ROS content in NG108-15 cells was significantly reduced after Mel intervention, indicating that Mel can reduce the excessive production of ROS caused by combined exposure to fluorine and aluminum, and Mel can alleviate the oxidative stress of NG108-15 cells caused by combined exposure to fluorine and aluminum.

[0089] (2) Exogenous supplementation of Mel increases ATP and mitochondrial membrane potential levels in NG108-15 cells exposed to fluoride and aluminum Synapses require a lot of energy to effectively transmit signals. Mitochondria are organelles located in eukaryotic cells, providing about 90% of the energy for cells in the form of ATP, and are responsible for some important pathways, including calcium homeostasis, apoptosis, cell signaling, oxidative stress, oxidative phosphorylation (OXPHOS), and participation in biosynthesis. Excessive mitochondrial fission can lead to imbalances in mitochondrial function and the occurrence of various diseases including neurodegenerative diseases. In order to evaluate the effect of Mel on ATP production in NG108-15 cells exposed to fluorine and aluminum, the present invention detected ATP levels.

[0090] The results are as follows Figure 6 As shown, compared with the control group, the ATP level of NG108-15 cells in the FA group was decreased, and the difference was statistically significant ( p <0.05); compared with the FA group, the ATP level of NG108-15 cells in the FA + Mel group increased, and the difference was statistically significant ( p <0.05).

[0091] The ATP level in NG108-15 cells increased significantly after Mel intervention, indicating that Mel could improve the reduction of ATP production caused by combined fluoride and aluminum exposure.

[0092] The present invention further evaluates the effect of Mel on the mitochondrial membrane potential of NG108-15 cells and detects the mitochondrial membrane potential level of NG108-15 cells.

[0093] The results are as follows Figure 7 As shown, compared with the control group, the mitochondrial membrane potential level of NG108-15 cells in the FA group decreased; compared with the FA group, the mitochondrial membrane potential level of NG108-15 cells in the FA + Mel group increased.

[0094] After Mel intervention, the mitochondrial membrane potential level in NG108-15 increased significantly, indicating that Mel can improve the decreased mitochondrial membrane potential level caused by combined exposure to fluorine and aluminum. Combined with the above-mentioned reduction of ROS content and improvement of ATP production by Mel, it shows that Mel can reverse the mitochondrial dysfunction caused by combined exposure to fluorine and aluminum.

[0095] 4. Exogenous supplementation of Mel inhibits mitochondrial fission in NG108-15 cells exposed to fluoride and aluminum Drp1 is a cytoplasmic protein that initiates mitochondrial fission by binding to Fis1 on the mitochondrial surface (the recruitment of mitochondrial Drp1 also requires the participation of MFF), leading to increased ROS generation and cell apoptosis. In order to evaluate the role of Mel in mitochondrial fission induced by combined fluoride and aluminum exposure, the present invention first preliminarily detected the expression of Drp1 and Fis1, the classic molecules of mitochondrial fission, at the transcriptional level.

[0096] The results are as follows Figure 8 As shown in Figures A and B, compared with the control group, the mRNA expressions of Drp1 and Fis1 in NG108-15 cells in the FA group were upregulated, and the difference was statistically significant ( p <0.05); compared with the FA group, the mRNA expression levels of Drp1 and Fis1 in NG108-15 cells in the FA + Mel group were downregulated, and the difference was statistically significant ( p <0.05).

[0097] The results showed that Mel reversed the promoting effect of combined fluoride and aluminum exposure on the expression of Drp1 and Fis1 mRNA in NG108-15 cells.

[0098] Based on the mRNA results of Drp1 and Fis1, the protein expression levels of Drp1, Fis1, and MFF in NG108-15 cells were further detected.

[0099] The results are as follows Figure 8 As shown in Figures C, D, E, and F, compared with the control group, the protein expressions of Drp1, Fis1, and MFF in NG108-15 cells in the FA group were upregulated, and the differences were statistically significant ( p <0.05); compared with the FA group, the protein expressions of Drp1, Fis1, and MFF in NG108-15 cells in the FA + Mel group were downregulated, and the difference was statistically significant ( p <0.05).

[0100] The results showed that, consistent with the protective effect of Mel on Drp1 and Fis1 mRNA in NG108-15 cells exposed to fluorine and aluminum, Mel could reverse mitochondrial fission in neurons induced by fluorine and aluminum exposure.

[0101] 5. Exogenous supplementation of Mel inhibits mitochondrial fission in NG108-15 cells exposed to fluorine and aluminum by upregulating the SIRT1 / PGC-1α pathway (1) Exogenous supplementation of Mel promotes the expression of SIRT1 / PGC-1α signaling pathway in NG108-15 cells exposed to fluoride and aluminum To evaluate the effect of Mel intervention on the SIRT1 / PGC-1α pathway, the mRNA expression of the SIRT1 / PGC-1α pathway was detected at the transcriptional level.

[0102] The results are as follows Fig. 9 As shown in Figures A and B, compared with the control group, the mRNA expression of SIRT1 in NG108-15 cells in the FA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA group, the mRNA of SIRT1 in NG108-15 cells in the FA + Mel group was upregulated, and the difference was statistically significant ( p <0.05); compared with the control group, the mRNA expression of PGC-1α in NG108-15 cells in the FA group and FA + Mel group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA group, the mRNA expression of PGC-1α in NG108-15 cells in the FA + Mel group was upregulated, and the difference was statistically significant ( p <0.05).

[0103] The results showed that Mel played a protective role in the suppression of SIRT1 / PGC-1α pathway mRNA expression caused by combined fluoride and aluminum exposure.

[0104] Based on the transcription results, the present invention further evaluates the protein expression level of the SIRT1 / PGC-1α pathway.

[0105] result Fig. 9 As shown in Figures C, D, and E, compared with the control group, the protein expressions of SIRT1 and PGC-1α in NG108-15 cells in the FA group and FA + Mel group were downregulated, and the differences were statistically significant ( p <0.05); compared with the FA group, the protein expressions of SIRT1 and PGC-1α in NG108-15 cells in the FA + Mel group were upregulated, and the difference was statistically significant ( p <0.05).

[0106] The results showed that Mel improved the inhibitory effect of fluoride and aluminum combined exposure on the expression of SIRT1 / PGC-1α pathway proteins in NG108-15 cells, indicating that Mel can reverse mitochondrial fission of neurons induced by fluoride and aluminum combined exposure by activating the SIRT1 / PGC-1α signaling pathway.

[0107] (2) Mel alleviates mitochondrial fission caused by combined fluoride and aluminum exposure by promoting the expression of PGC-1α After intervening NG108-15 cells with fluorine-aluminum combined exposure with PGC-1α siRNA, the changes in Drp1, a mitochondrial fission index, were detected to determine whether PGC-1α is one of the mechanisms by which Mel regulates mitochondrial fission. Fig.10 As shown:

[0108] ① Regarding the mRNA expression of PGC-1α, compared with the FA + Control siRNA group, the mRNA expression of PGC-1α in NG108-15 cells in the FA + Mel + Control siRNA group was upregulated, and the difference was statistically significant ( p <0.05), the mRNA expression of PGC-1α in NG108-15 cells in the FA + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel +Control siRNA group, the mRNA expression of PGC-1α in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p<0.05); compared with the FA + PGC-1α siRNA group, the mRNA expression of PGC-1α in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05).

[0109] ② Regarding the mRNA expression of Drp1, compared with the FA + Control siRNA group, the mRNA expression of Drp1 in NG108-15 cells in the FA + Mel + Control siRNA group was downregulated, and the difference was statistically significant ( p <0.05); the mRNA expression of Drp1 in NG108-15 cells in the FA + PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel +Control siRNA group, the mRNA expression of Drp1 in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05); compared with the FA + PGC-1α siRNA group, the mRNA expression of Drp1 in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p <0.05).

[0110] ③ Regarding the protein expression of PGC-1α, compared with the FA + Control siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel + Control siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + PGC-1α siRNA group and the FA + Mel + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + PGC-1α siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + Mel +PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05).

[0111] ④For the protein expression of Drp1, compared with the FA + Control siRNA group, the protein expression of Drp1 in NG108-15 cells in the FA + Mel + Control siRNA group was down-regulated, and the protein expression of Drp1 in NG108-15 cells in the FA + PGC-1α siRNA and FA + Mel + PGC-1α siRNA groups was up-regulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel +Control siRNA group, the protein expression of Drp1 in NG108-15 cells in the FA + PGC-1α siRNA and FA + Mel + PGC-1α siRNA groups was upregulated, and the difference was statistically significant ( p <0.05); compared with the FA + PGC-1α siRNA group, the protein expression of Drp1 in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was downregulated, and the difference was statistically significant ( p <0.05).

[0112] The results showed that Mel alleviated mitochondrial fission in NG108-15 cells induced by combined exposure to fluorine and aluminum by promoting the expression of PGC-1α, thereby protecting mitochondria.

[0113] To evaluate the effect of PGC-1α on Mel's improvement of mitochondrial homeostasis and cell damage in NG108-15 cells exposed to fluorine and aluminum, the ROS level in NG108-15 cells exposed to fluorine and aluminum was detected after intervention with PGC-1α siRNA.

[0114] The results are as follows Fig.11 As shown, compared with the FA + Control siRNA group, the ROS expression of NG108-15 cells in the FA + Mel + Control siRNA group was downregulated, and the difference was statistically significant ( p <0.05), the expression of ROS in NG108-15 cells in the FA + PGC-1α siRNA group and the FA + Mel + PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel + Control siRNA group, the expression of ROS in NG108-15 cells in the FA + Mel + PGC-1α siRNA group was upregulated, and the difference was statistically significant ( p <0.05); compared with the FA + PGC-1α siRNA group, the ROS expression of NG108-15 cells in the FA + Mel + PGC-1α siRNA group was downregulated, and the difference was statistically significant (p <0.05).

[0115] The results showed that Mel alleviated cellular oxidative stress caused by combined fluoride and aluminum exposure and reduced mitochondrial damage by promoting the expression of PGC-1α.

[0116] (3) Mel alleviates the inhibitory effect of fluoride and aluminum combined exposure on PGC-1α by promoting SIRT1 expression After intervention with SIRT1 siRNA, the protein expression level of PGC-1α in NG108-15 cells exposed to fluorine and aluminum was detected to determine whether SIRT1 is an upstream molecular event in the regulation of PGC-1α by Mel. Fig.12 As shown, we can find that:

[0117] ① Regarding the protein expression of SIRT1, compared with the FA + Control siRNA group, the SIRT1 protein expression of NG108-15 cells in the FA + Mel + Control siRNA group was upregulated, and the difference was statistically significant ( p <0.05), SIRT1 protein expression was downregulated in the FA + SIRT1 siRNA group, and the difference was statistically significant ( p <0.05). Compared with the FA + Mel + Control siRNA group, the protein expression of SIRT1 in NG108-15 cells in the FA + SIRT1 siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + SIRT1 siRNA group, the protein expression of SIRT1 in NG108-15 cells in the FA + Mel + SIRT1 siRNA group was upregulated, and the difference was statistically significant ( p <0.05).

[0118] ② Regarding the protein expression of PGC-1α, compared with the FA + Control siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + SIRT1 siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + Mel + Control siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + SIRT1 siRNA group was downregulated, and the difference was statistically significant ( p <0.05); compared with the FA + SIRT1 siRNA group, the protein expression of PGC-1α in NG108-15 cells in the FA + Mel + SIRT1 siRNA group was upregulated, and the difference was statistically significant ( p <0.05).

[0119] The results showed that Mel alleviated the inhibitory effect of fluorine-aluminum combined exposure on PGC-1α expression in NG108-15 cells by promoting the expression of SIRT1, indicating that SIRT1 is an upstream molecular event in the regulation of PGC-1α by Mel.

[0120] In summary, Mel can improve learning and memory disorders caused by neuronal cell damage or mitochondrial fission caused by combined exposure to fluorine and aluminum by promoting the SIRT1 / PGC-1α pathway, inhibiting mitochondrial fission, increasing ATP production and mitochondrial membrane potential levels, downregulating ROS levels, improving cell viability, improving mitochondrial dysfunction caused by combined exposure to fluorine and aluminum, and promoting the expression of synaptic proteins GAP-43 and Ng.

[0121] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as those in the embodiments, in order to avoid redundancy, the present invention describes a preferred embodiment. Although the preferred embodiments of the present invention have been described, once those skilled in the art know the basic inventive concept, they can make other changes and modifications to these embodiments, and these changes and modifications all fall within the scope of the present invention.

[0122] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the equivalent technology of the present invention, the present invention is also intended to include these modifications and variations.

Claims

1. The application of melatonin in the preparation of drugs for treating learning and memory disorders caused by nerve cell damage or mitochondrial fission.

2. The use according to claim 1, characterized in that: The melatonin is used for preparing medicine for treating nerve cell damage caused by combined exposure to fluorine and aluminum or learning and memory disorders caused by mitochondrial fission.

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

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

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

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

7. The use according to claim 1, characterized in that: The auxiliary material is one or any combination of fillers, disintegrants, lubricants, suspending agents, adhesives, sweeteners, flavoring agents, preservatives, bases, and antioxidants.

8. The use according to claim 1, characterized in that: The melatonin is used for preparing an expression promoter of synaptic protein GAP-43 or Ng.

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