Application of walnut peptide in preparation of medicine for improving and treating neurodegenerative diseases induced by aluminum exposure

Abstract

The invention discloses application of walnut peptide in preparation of medicine for improving and treating neurodegenerative diseases induced by aluminum exposure, and relates to the technical field of biology. A rat model is established by intraperitoneal injection of aluminum maltol and intragastric administration of walnut peptide; it is proved that the walnut peptide can inhibit aluminum exposure induced mi R-124-3p-STAT3-TGF-beta1 / Smad3 protein increase, the walnut peptide has an effective neuroprotective effect on learning and memory defects in a sub-chronic aluminum maltol exposure rat model, and therefore the walnut peptide can be used for preparing the medicine for preventing and treating the sub-chronic aluminum maltol exposure rat model from suffering from the neuroprotective effect, and the walnut peptide can be used for preparing the medicine for preventing and treating the sub-chronic aluminum maltol exposure rat model from suffering from the neuroprotective effect and preventing and treating the sub-chronic aluminum maltol exposure rat model from suffering from the neuroprotective effect. The walnut peptide has potential application value in preparation of drugs for improving and treating neurodegenerative diseases induced by aluminum exposure.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to the application of walnut peptide in preparing medicines for improving and treating neurodegenerative diseases induced by aluminum exposure. Background Art

[0002] Aluminum is the third most abundant element in the earth's crust, second only to oxygen and silicon, and is the most abundant metallic element. It is widely used in military, industrial, medical and human life, such as aluminum-based batteries, drinking water treatment systems, kitchen utensils, drugs and antiperspirants, and can be absorbed by the human body through a series of pathways. Excessive intake of aluminum accumulates in several tissues and organs and is difficult to excrete quickly, especially in the brain. Aluminum is mainly deposited in the hippocampus, and long-term accumulation can lead to cognitive dysfunction, as evidenced by significant learning and memory impairment, which is closely related to many neurodegenerative diseases such as Alzheimer's disease (AD). Neurodegenerative diseases are diseases involving the loss of neurons or myelin and progressive neuronal dysfunction. Aluminum is neurotoxic, which is related to the accumulation of aluminum in the hippocampus, cortex and other sensitive areas.

[0003] We found that aluminum exposure may lead to decreased miR-124-3p levels, increased STAT3, TGF-β1 and Smad3 protein levels, and neuronal apoptosis. The intervention of walnut peptides can slow down the expression of the above genes and proteins. In view of this, the inventors proposed the use of walnut peptides in the preparation of drugs for improving and treating aluminum exposure-induced neurodegenerative diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a new application of walnut peptides for preparing medicines for improving and treating neurodegenerative diseases induced by aluminum exposure.

[0005] The above technical purpose of the present invention is achieved through the following technical scheme: application of walnut peptide in the preparation of medicines for improving and treating neurodegenerative diseases induced by aluminum exposure.

[0006] The present invention is further configured as follows: the drug is a drug that inhibits the decrease of miR-124-3p and the increase of STAT3, TGF-β1, and Smad3 proteins induced by aluminum exposure.

[0007] The present invention is further configured as follows: the medicine is a preparation prepared by taking walnut peptide as an active ingredient and adding pharmaceutically acceptable excipients.

[0008] The present invention is further configured as follows: the dosage form of the preparation is an injection.

[0009] In summary, the present invention has the following beneficial effects: the present invention establishes a rat model of long-term low-level aluminum exposure, and proves that subchronic aluminum exposure induces changes in the miR-124-3p-STAT3-TGF-β1 / Smad3 pathway in rat hippocampal tissue, and confirms that walnut peptide can inhibit the decrease of miR-124-3p and the increase of STAT3, TGF-β1, and Smad3 proteins induced by aluminum exposure. Walnut peptide has an effective neuroprotective effect on learning and memory defects in the subchronic maltol aluminum exposure rat model. Therefore, walnut peptide has potential application value in the preparation of drugs for improving and treating neurodegenerative diseases induced by aluminum exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 This is a graph showing the effect of walnut peptide on the relative brain weight of rats exposed to maltol aluminum poisoning in the present invention (n=10. a P<0.05, aa P<0.01vs model group; b P<0.05, bb P<0.01vs treatment group; c P<0.05, cc P < 0.01 vs prevention group);

[0011] Figure 2 The Morris water maze test diagram of rats in the embodiment of the present invention (wherein, (A) the escape latency of each group of rats; (B) the number of times each group crossed the platform; (C) the frequency of rats crossing the platform quadrant; n = 10. a P<0.05, aa P<0.01vs model group; b P<0.05, bb P<0.01vs treatment group; c P<0.05, cc P < 0.01 vs prevention group);

[0012] Figure 3 is a trajectory diagram of a rat water maze in an embodiment of the present invention;

[0013] Figure 4 The invention discloses an application of walnut peptide in improving and treating aluminum exposure-induced neuronal apoptosis observed by transmission electron microscopy.

[0014] Figure 5 This is a diagram showing the effect of walnut peptides on corresponding proteins in rats exposed to aluminum maltol in an example of the present invention. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] Embodiment 1:

[0017] Drugs and reagents

[0018] Aluminum trichloride (crystalline) (AlCl3·6H2O, purity 97%, Tianjin Fengchuan Chemical Reagent Co., Ltd.); maltol (analytical grade, 99% purity, Sigma-Aldrich, USA); walnut peptide (purity 98%, small peptide); phosphate buffered saline (PBS), 0.90% sodium chloride solution and sodium pentobarbital (Shandong Chenxin Pharmaceutical Co., Ltd.); chloroform, isopropanol, anhydrous ethanol; total RNA extractor (Trizol) (B511311-0025), M-MuLV first-strand cDNA synthesis kit (B532435), 2Xsg Fast qPCR Master Mix (B639271) (Shanghai Bioengineering); RIPA strong lysis buffer CW2333S, protease inhibitors, phosphatase inhibitors, BCA protein quantification kit CW0014S (Beijing Kangwei Century Biotechnology Co., Ltd.); Rainbow 180 broad-spectrum protein marker PR1910, bovine serum albumin (BSA, Beijing Solebold Technology Co., Ltd.); electrophoresis, transmembrane solution, TBS (Wuhan Sewell Biotechnology Co., Ltd.); SDS-PAGE preparation kit (Shanghai Yazyme); anti-GADPH antibody (ERP16891), anti-STAT3 antibody (ERP787Y), anti-TGFβ1 antibody (ERP21143), anti-Smad3 antibody (EP568Y), goat anti-rabbit IgG HL (HRP) (Abcam, UK); phospho-SMAD3 (Ser423) polyclonal antibody (PA5-106154), phospho-Smad3 (Ser425) polyclonal antibody (PA5-104939), phospho-STAT3 (Ser727) polyclonal antibody (PA5-17876), phospho-STAT3 (Tyr705) monoclonal antibody (r.263.6) (MA5-15193) (Thermo Fisher Scientific, USA); centrifuge (Shanghai Aiyan Biotechnology Co., Ltd.); chemiluminescence imaging system (Gelpro 32); and decolorization shaker (Beijing Liuyi Instrument Factory).

[0019] Preparation of maltoaluminum

[0020] In 2011, JECFA revised the permissible weekly aluminum intake to 2 mg / kg. Using the equivalent dose ratio derived from the body surface area conversion between humans and animals, and the dose conversion between different routes of administration, the daily aluminum intake of an adult weighing 60 kg does not exceed 0.283 mg / kg, and the intraperitoneal aluminum exposure dose of rats is 0.535 mg / kg. Based on the experience of previous experiments, the contamination dose was set at 20 μmol / kg, and the control group used an equal volume of normal saline. Prepare a 40 mmol / L aluminum chloride solvent by mixing AlCl3·6H2O in saline, and dissolve maltol in PBS to prepare a 120 mmol / L maltol solvent, then mix the 40 mmol / L aluminum chloride solvent with the 120 mmol / L maltol solvent in equal volumes to obtain the final 20 mmol / L maltol aluminum solution.

[0021] Preparation of walnut peptide

[0022] The walnut peptide powder was dissolved in sterile ultrapure water to obtain an 800 mg / kg walnut peptide solution.

[0023] Grouping and housing of experimental animals

[0024] Thirty-two 6-week-old SPF-grade healthy male SD rats (purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.), weighing 190±20g, were kept in an animal house, the temperature was maintained at 22℃-26℃, the humidity was maintained at 45%-50%, the dark-light cycle was 12 hours (8:00-20:00), and they were fed with ordinary feed and could eat and drink water. After 5 days of adaptive feeding, the animals were randomly divided into groups according to their weight: control group, poisoning group, treatment group and prevention group. Maltol aluminum was intraperitoneally injected for staining from Monday to Friday, 1ml / kg was administered, and no staining was performed on Saturdays and Sundays for 90 consecutive days. The control group was injected with an equal amount of normal saline. The treatment group and the prevention group began to be gavaged with walnut peptides on the 45th day and the 1st day of infection, respectively.

[0025] Experimental Example 1:

[0026] The rats were weighed and recorded every other week, and the mental state and growth of the rats as well as the healing degree of the intraperitoneal injection needle incision were observed every day.

[0027] Experimental results:

[0028] In the control group, rats showed healthy mental state, with soft fur, soft abdomen, and normal eating and drinking conditions. In the prevention group, rats showed improved mental state, with soft fur, slightly hard abdomen, and better eating and drinking conditions. In the treatment group, rats showed signs of mental distress, with rough fur, hard abdomen, and reduced activity. In the poisoning group, rats showed signs of mental distress, reduced activity, rough fur, and hard abdomen.

[0029] Throughout the experimental period, the relative brain-to-body weight of rats in all groups changed to reflect normal growth (F=98.537, P<0.001). Compared with the control group, the relative brain weight of rats in the maltol aluminum exposure group was found to be reduced (P<0.01). Compared with the model group, the relative brain weight of rats in the walnut peptide treatment group and the prevention group gradually increased (P<0.01). The decrease in the brain-to-body weight ratio of aluminum exposure may be related to the decrease in the number of neuronal cells. Figure 1 shown.

[0030] Experimental Example 2:

[0031] Morris water maze test

[0032] 2.1 Experimental steps:

[0033] (1) Morris water maze setup: The pool was evenly divided into four quadrants, with the platform located in one quadrant. The water temperature was maintained between 22°C and 24°C, the curtains were drawn, and the environment was consistently set to be quiet. Before the formal experiment, the rats were allowed to swim freely for 120 seconds to familiarize themselves with the platform, which was about 4 cm below the water surface. For 5 consecutive days before the start of the experiment, the rats were placed in the water from different quadrants directly opposite the pool wall. The time and path taken by the rats from entering the water to finding the platform were recorded. The average time required to find the platform across the four quadrants was calculated as the escape latency. In the event that the rats failed to find the platform within 120 seconds, they were manually guided to the platform and allowed to stay on the platform for 10 seconds, with 120 seconds being the recording time.

[0034] (2) Spatial exploration: On the 6th day, after the platform was removed, the rats were placed in the water from the designated quadrant, and the number of times they crossed the original position of the platform and the platform quadrant was counted within 120 seconds.

[0035] 2.2 Experimental results:

[0036] The results showed that in the positioning cruising experiment, the escape latency of each group of rats on the first and second days was significantly different only in the maltol aluminum exposure model group compared with the control group (P<0.01). On the third, fourth, and fifth days, the average escape latency of rats exposed to maltol aluminum showed an upward trend compared with the control group, and the difference was statistically significant (P<0.01). Among them, compared with the maltol aluminum exposed rats in the model group, the average escape latency of rats in the walnut peptide treatment group and the prevention group showed a downward trend, and the difference was statistically significant (P<0.01). In particular, on the fourth and fifth days, the difference in the average escape latency of rats between the walnut peptide treatment group and the prevention group was statistically significant (P<0.01). This shows that long-term low-level aluminum exposure destroys the learning ability of rats, and the intervention of walnut peptides can significantly improve the learning ability deficit caused by aluminum exposure.

[0037] On the sixth day of the spatial exploration test, the frequency of crossing the platform and platform quadrants in all maltol aluminum exposure groups decreased compared with the counts in the control group (P<0.05), especially compared with the maltol aluminum exposure model group and the walnut peptide treatment group, the number of times the rats in the walnut peptide prevention group crossed the platform quadrant increased, and this difference was statistically significant (P<0.05). Figure 2 and 3 This finding indicates that long-term low-level exposure to aluminum leads to deficits in spatial memory and cognitive impairment in rats, confirming that aluminum can impair learning, memory, and cognitive abilities, and that walnut peptides can reverse the behavioral impairments of subchronic aluminum exposure.

[0038] Experimental Example 3:

[0039] Observation of apoptosis of neurons in rat brain tissue by transmission electron microscopy

[0040] 3.1 Experimental steps:

[0041] Rats were killed after deep anesthesia, and the brain tissue of rats was quickly removed according to the established experimental protocol. Careful operation was performed to avoid damaging the brain tissue. The cortex was separated from the hippocampus, and both ends of the hippocampal tissue were removed, leaving only a small piece of tissue about 0.5 cm wide in the middle of the hippocampus. It was rinsed with distilled water for 2-3 seconds to remove the residual blood on the surface. It was fixed in a pre-cooled fixative (2% glutaraldehyde) with a toothpick and stored in a 4% refrigerator. The tissue was rinsed with buffer for 2-3 hours, during which the solution was changed 4 times, fixed with osmium acid (1% OSO4) for no more than 2 hours, and each piece was dehydrated in acetone for 5-15 minutes in series, immersed in a mixture of embedding medium and acetone at room temperature for 2-4 hours, and then embedded in epoxy resin, immersed at 40°C for 24 hours, and hardened at 60°C for 48 hours. LKB ultrathin slicer; uranyl acetate, lead citrate, repeated staining for 15-30 minutes each time; transmission electron microscopy observation.

[0042] 3.2 Experimental results:

[0043] Transmission electron microscopy was used to observe neuronal apoptosis in rat brain tissue (indicated by red arrows), which showed that the hippocampal neurons in the control group showed typical morphology and structure, with complete nuclear structure; the nuclei of hippocampal neurons in the maltol aluminum poisoning model group had dense adhesive chromatin and nuclear condensation; the walnut peptide treatment group had dense chromatin and nuclear shrinkage; the walnut peptide prevention group had normal morphology and structure of hippocampal neurons; aluminum exposure can cause apoptosis of rat hippocampal neurons. Figure 4 shown.

[0044] Experimental Example 4:

[0045] Detection of the expression levels of miR-124-3p, mstat3, mTGF-β1, msmad3, mBcl-2, and mCaspase3 in rat hippocampal tissue

[0046] 4.1 Experimental methods:

[0047] (1) RT-qPCR detection of miR-124-3p and its mRNA expression: A certain amount of cerebral cortex tissue was weighed and ground with 1 mL of Trizol on ice to isolate total RNA from the cerebral cortex tissue. U6 was used as the endogenous control for miR-124-3p, and GAPDH was used as the endogenous control for mRNA. Separate primer sets were designed for miR-124-3p, mRNA, and their respective internal controls. RNA was converted into cDNA according to the reverse transcription protocol, and then cDNA was amplified using it as a template according to the instructions of the kit. The PCR conditions were set as follows: initial denaturation at 95°C for 15 minutes; followed by denaturation at 95°C for 34 seconds and annealing at 60°C for 34 seconds. The relative expression levels of genes were determined using the 2-δδCt method. The gene sequences are shown in Table 1.

[0048] 4.2 Experimental results:

[0049] (1) The results showed that compared with the control group, the relative expression levels of miR-124-3p and mBcl-2 in rats exposed to maltoaluminum were decreased (P<0.05). Compared with the model group, the relative expression levels of miR-124-3p and mBcl-2 as well as the relative expression levels of miR-124-3p, mBcl-2 and mBax in the rats in the walnut peptide treatment group and the prevention group were gradually decreased (P<0.05). Compared with the control group, the relative expression levels of mstat3, mTGF-β1, msmad3 and mCaspase3 in rats in each group exposed to maltol aluminum increased (P<0.05); compared with the model group, the relative expression levels of mstat3, mTGF-β1, msmad3 and mCaspase3 in rats in the walnut peptide treatment group and the prevention group gradually decreased (P<0.05), and this difference was statistically significant. See Table 1 and Figure 5 The above results indicate that exposure to maltol aluminum can cause neuronal apoptosis. The apoptosis in the walnut peptide intervention group was less affected than that in the pollution group, suggesting that walnut peptide can alleviate or repair apoptosis to a certain extent.

[0050] Table 1 Primer sequences

[0051]

[0052]

[0053] Test Example 5:

[0054] Western blot was used to detect the expression of p-STAT3 Ser727, p-STAT3 Tyr705, STAT3, p-Smad3 Ser423, p-Smad3 Ser425, Smad3 and TGF-β1 proteins in rat hippocampus tissue

[0055] 5.1 Experimental methods:

[0056] After killing the rats, the hippocampus and cortical regions of the rat brain were separated. The hippocampus tissue was weighed and the RIPA lysis buffer was added, and the supernatant was centrifuged to extract the protein and measure the concentration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), 80V electrophoresis was performed until the band ran out of the concentrated gel, and the voltage was increased to 120V. After the end, the resolved protein was transferred to a PVDF membrane. After the membrane was transferred, the PVDF membrane was immersed in a blocking solution containing 5% skimmed milk powder, incubated at room temperature for 2 hours, and then rinsed with TBST. Then, primary antibodies (p-STAT3 Ser727, p-STAT3 Tyr705, STAT3, p-Smad3 Ser423, p-Smad3 Ser425, Smad3 and TGF-β1) were added at a dilution ratio of 1:1000; GADPH was loaded at a dilution of 1:2000, the membrane was incubated at 4°C overnight for 18 h, and then washed three times with TBST, horseradish peroxidase-labeled goat anti-mouse antibody and goat anti-rabbit antibody were added (dilution of 1:4000), the membrane was washed and ECL color was developed, and the color was exposed in a dark room. The ratio of the gray value of the target protein to the internal standard GAPDH represented the expression level of the target protein, and Gelpro32 statistical analysis was performed.

[0057] 5.2 Experimental Results

[0058] The data showed that when compared with the control group, the relative expression levels of p-STAT3 Ser727, p-STAT3 Tyr705, TGF-β1, p-Smad3 Ser423, and p-Smad3 Ser425 increased in all groups of rats exposed to maltoaluminum (P<0.05); compared with the model group, the relative expression levels of P-stat 3 ser 727, p-STAT3Tyr705, TGF-β1, p-Smad3 Ser423, and p-Smad3 Ser425 in the walnut peptide treatment group and the prevention group gradually decreased (P<0.05), and this difference was statistically significant. Figure 5 Walnut peptides alleviated and reversed the damage of maltol aluminum to the rat nervous system. Walnut protein contains active peptides with antioxidant effects. This may be because walnut peptides inhibited the redox imbalance induced by aluminum, thereby inhibiting the increase in protein levels and reducing oxidative damage to lipids and proteins.

[0059] In summary, this study established a rat model of long-term low-level aluminum exposure, and demonstrated that subchronic aluminum exposure induced changes in the miR-124-3p-STAT3-TGF-β1 / Smad3 pathway in rat hippocampus tissue, and walnut peptides were able to inhibit the decrease of miR-124-3p and the increase of STAT3, TGF-β1, and Smad3 proteins induced by aluminum exposure. Walnut peptides have an effective neuroprotective effect on learning and memory deficits in the subchronic maltol aluminum exposure rat model.

[0060] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of walnut peptides in the preparation of medicines for improving and treating neurodegenerative diseases induced by aluminum exposure.

2. The use of the walnut peptide according to claim 1 in the preparation of a medicine for improving and treating aluminum exposure-induced neurodegenerative diseases, characterized in that: The drug is a drug that inhibits the decrease of miR-124-3p and the increase of STAT3, TGF-β1, and Smad3 proteins induced by aluminum exposure.

3. The use of the walnut peptide as claimed in claim 1 in the preparation of a medicine for improving and treating neurodegenerative diseases induced by aluminum exposure, characterized in that: The medicine is a preparation prepared by taking walnut peptide as an active ingredient and adding pharmaceutically acceptable auxiliary materials.

4. The use of the walnut peptide as claimed in claim 3 in the preparation of a medicine for improving and treating neurodegenerative diseases induced by aluminum exposure, characterized in that: The dosage form of the preparation is injection.

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