Use of methyl palmitate in the preparation of a medicament for the treatment and / or prevention of aging and related cognitive impairment

Abstract

The application discloses application of methyl palmitate in preparation of a medicine for treating and / or preventing senility and related cognitive impairment, and belongs to the technical field of biological medicine. The application proves by a cell experiment that methyl palmitate can significantly inhibit D-galactose-induced SH-SY5Y cell senility, and effectively reduce the expression level of senility-related proteins P53 and P16. The application proves by a behavior experiment that methyl palmitate can significantly improve the cognitive index of a senility model mouse, shorten an escape latency, and effectively improve learning ability reduction, spatial memory decline and cognitive function impairment caused by senility. In addition, methyl palmitate can significantly enhance the long-term potentiation effect of the hippocampal CA1 region of the senility model mouse, and reverse senility-related synaptic transmission damage. The application discloses the application of methyl palmitate in resisting neural senility and improving senility-related cognitive impairment, and provides a new candidate molecule for preparing a medicine for treating neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the use of methyl palmitate in the preparation of drugs for the treatment and / or prevention of aging and related cognitive impairment. Background Technology

[0002] Aging is an irreversible and progressive decline in bodily functions that occurs with age. Nervous system aging can lead to cognitive decline, resulting in various neurodegenerative diseases such as Alzheimer's and Parkinson's. With the accelerating aging of the global population, age-related cognitive impairments, such as memory loss, decreased learning ability, and impaired executive function, have become a major public health problem seriously threatening the quality of life of the elderly. Therefore, developing drugs that can delay neuroaging and improve cognitive function has significant social and economic value.

[0003] Methyl palmitate (MP) is a naturally occurring fatty acid ester widely found in various plants. Its molecular structure combines a polar carboxyl group with nonpolar aliphatic carbons, exhibiting good safety for organisms. Existing research indicates that methyl palmitate possesses various pharmacological activities, including antioxidant, anti-inflammatory, anti-fibrotic, and anti-apoptotic effects. However, whether methyl palmitate has anti-neuroaging effects and whether it can be used to improve age-related cognitive impairment has not yet been reported. Summary of the Invention

[0004] In view of this, the present invention provides the use of methyl palmitate in the preparation of drugs for the treatment and / or prevention of aging and related cognitive impairment. Animal experiments and cell-level experiments have shown that methyl palmitate has anti-aging effects and improves cognitive and learning memory abilities, providing a novel candidate drug for the treatment of neurodegenerative diseases.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides the use of methyl palmitate in the preparation of medicaments for the treatment and / or prevention of aging and related cognitive impairment.

[0006] Furthermore, the molecular formula of the methyl palmitate is CH3(CH2). 14 CO2CH3, CAS number: 112-39-0, chemical structural formula: .

[0007] Furthermore, methyl palmitate is used as the sole active ingredient in this application.

[0008] Furthermore, the methyl palmitate inhibits the expression levels of aging-related proteins P53 and P16.

[0009] Furthermore, the methyl palmitate can enhance the long-term potentiation of the hippocampus.

[0010] In a second aspect, the present invention provides a medicament for preventing aging and related cognitive impairment, the medicament comprising methyl palmitate and medically acceptable pharmaceutical excipients.

[0011] Furthermore, the methyl palmitate is the sole active ingredient.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention reveals the application of methyl palmitate in the preparation of drugs for the treatment and / or prevention of aging and related cognitive impairment, providing a new candidate drug for the intervention of neurodegenerative diseases. Cellular experiments confirmed that methyl palmitate can significantly inhibit D-galactose-induced senescence of SH-SY5Y cells and effectively reduce the expression levels of aging-related proteins P53 and P16. Behavioral experiments confirmed that methyl palmitate can significantly improve the cognitive index and shorten the escape latency in aging model mice, effectively improving age-related learning decline, spatial memory impairment, and cognitive dysfunction. Furthermore, methyl palmitate can significantly enhance the long-term potentiation effect in the CA1 region of the hippocampus in aging model mice, effectively reversing age-related damage to synaptic transmission. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below.

[0014] Figure 1 The figure shows the effect of different doses of MP on the expression of P53 and P16 proteins in a D-Gal-induced cell senescence model. Figure 1 A is a Western blot image of protein bands. Figure 1 B represents the quantitative analysis of protein expression.

[0015] Figure 2 This is a graph showing the effect of MP on D-Gal-induced cognitive function in aging mice. Figure 2 A represents the cognitive index of the new object recognition experiment. Figure 2 B represents the incubation period for escaping the water maze. Figure 2 C represents the number of times the platform was traversed. Figure 2 D represents the average swimming speed.

[0016] Figure 3 This is a diagram showing the effect of MP on D-Gal-induced hippocampal LTP in aging mice. Figure 3 A is a scatter plot of LTP. Figure 3 B is the LTP statistics chart. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. It should be noted that, unless otherwise specified, all chemical reagents involved in this invention are purchased through commercial channels.

[0018] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. It should be noted that the terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the exemplary embodiments of this application.

[0019] Source of methyl palmitate: The methyl palmitate used was purchased from McLean Company (product number: M813485).

[0020] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0021] Example 1: Effect of MP on a D-galactose-induced senescence model of SH-SY5Y cells I. Experimental Methods 1. Cell Culture SH-SY5Y cells were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotics, and incubated in a constant temperature incubator at 37°C and 5% CO2. When the cells grew to about 80%-90% confluence, they were passaged by digestion with 0.25% trypsin-EDTA.

[0022] 2. Grouping and Processing Log-phase SH-SY5Y cells were seeded into 6-well plates at a density of 3 × 10⁶ cells per well. 5 After culturing for 24 hours to allow the cells to fully adhere, the following grouping and treatment were performed: (1) Blank control group: Replace with conventional complete culture medium and continue culturing for 96 hours; (2) Model group: The culture medium was replaced with a complete medium containing 35 g / L D-galactose (D-Gal) to induce cell senescence model and cultured for another 96 hours; (3) Drug treatment group: The culture medium was replaced with complete medium containing 35 g / L D-Gal and different concentrations of MP. Four concentration gradients of MP were set: 50 μM, 100 μM, 200 μM and 500 μM. The cells were incubated with D-Gal for 96 hours. The drug MP was prepared as a high-concentration stock solution using dimethyl sulfoxide (DMSO) and stored at -20°C in the dark. During the experiment, the stock solution was diluted with complete culture medium to the required working concentration to ensure that the final concentration of DMSO in each treatment group was consistent and did not exceed 0.1% (v / v). This concentration had no significant effect on cell viability.

[0023] Each group should have at least 3 replicates, and all experiments should be repeated 3 times independently.

[0024] 3. Western Blot After processing, the culture supernatant was discarded, and the cells were gently washed twice with pre-cooled phosphate-buffered saline (PBS). Then, according to the requirements of subsequent detection indicators (Western blotting to detect P16 and P53 protein expression levels), the appropriate cell lysis buffer was added, and the cells were lysed on ice to collect whole protein samples for subsequent analysis. After protein denaturation, SDS-polyacrylamide gel electrophoresis was performed, followed by transfer of the protein to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 2 hours, washed with TBST, and incubated overnight at 4°C with primary antibody. GAPDH was used as an internal control. The next day, the membrane was washed three times with TBST, incubated with the appropriate secondary antibody for 2 hours, and then washed three more times with TBST. Finally, images were acquired and saved using ECL chemiluminescence imaging analysis system.

[0025] II. Experimental Results This embodiment examines the effect of MP on the expression of aging markers P16 and P53 proteins in a D-Gal-induced neural cell senescence model at the in vitro cellular level. The results are as follows: Figure 1 As shown, compared with the blank control group, the expression levels of P53 and P16 proteins in the model group were significantly increased, indicating that D-Gal successfully induced senescence in SH-SY5Y cells. Compared with the model group, the expression levels of P53 and P16 proteins decreased to varying degrees after treatment with different concentrations of MP, showing a certain dose-dependent effect. Among them, the inhibitory effects of 200 μM and 500 μM MP treatment groups were more significant, indicating that methyl palmitate can effectively inhibit the expression levels of senescence-related proteins P16 and P53 in the D-Gal cell senescence model.

[0026] Example 2: Effects of MP on learning and memory abilities in D-Gal-induced aging mice I. Experimental Materials and Methods 1. Establishment of aging models SPF-grade C57BL / 6 mice (36 mice in total, 12 mice in each group), 8 weeks old and weighing 20-25g, were used. They were acclimatized for 7 days under standard conditions (temperature 22±2℃, 12h light / dark cycle) with free access to food and water. The C57BL / 6 mice were divided into three groups according to weight and sex: blank control group, model group, and drug treatment group. The blank control group was injected intraperitoneally with physiological saline every day, while the other two groups of mice were injected intraperitoneally with an equal volume of 200mg / kg D-Gal solution every day for 8 consecutive weeks.

[0027] 2. Grouping and Processing After successful model establishment, drug administration is performed as follows: Blank control group: Daily gavage administration of an equal volume of DMSO solution for 8 consecutive weeks; Model group: Daily gavage administration of an equal volume of DMSO solution for 8 consecutive weeks; Drug treatment group: MP 300 mg / kg was administered by gavage daily for 8 weeks.

[0028] 3. New Object Recognition Experiment The new object recognition experiment was divided into an adaptation phase, a familiarization phase, and a testing phase. In the adaptation phase, each mouse was placed individually in an empty space box and allowed to explore freely for 10 minutes to familiarize itself with the experimental environment and reduce stress response. In the familiarization phase, the mice were allowed to come into contact with and memorize two identical objects (A1 and A2) and explore freely for 10 minutes. In the testing phase, one of the old objects (A2) was replaced with a new object (B), and the mice were allowed to explore freely for 10 minutes. The exploration time of the mice for the new object and the old object was recorded.

[0029] The Recognition Index (RI) is calculated as follows: RI = Time spent exploring new objects / (Time spent exploring new objects + Time spent exploring old objects). 4. Morris water maze experiment The Morris water maze test is used to assess the spatial learning and memory abilities of mice. It mainly consists of a circular pool, a platform, a tracking system, and analysis software. The pool is 120 cm in diameter, with a water depth maintained at 70-90 cm and a water temperature controlled at 21±1℃ to ensure the mice are in a suitable physiological state. The platform is made of transparent material, approximately 10 cm in diameter, and its height is slightly below the water surface by 1-2 cm, preventing the mice from directly observing it but allowing them to reach it by swimming.

[0030] In each training session, mice were randomly placed into the water from three different quadrants and allowed to swim freely, searching for a platform hidden beneath the surface. If a mouse found a platform within 90 seconds, it was allowed to stay on the platform for 10 seconds, and the latency was recorded. If the mouse failed to find the platform within the allotted time, it was guided to stay on the platform for the same amount of time, and the latency was recorded as 90 seconds. The training was conducted for 5 consecutive days. The experiment was paused on the 6th day, and the test was conducted on the 7th day. During the test, the platform was removed, and parameters such as the time it took for the mouse to find the original platform (i.e., the escape latency), the number of times it crossed the original platform, and its swimming speed were recorded.

[0031] II. Experimental Results This embodiment uses behavioral experiments to comprehensively evaluate the effect of MP on improving the learning and memory abilities of D-Gal-induced aging mice. The results are as follows: Figure 2 As shown. In the new object recognition experiment, as... Figure 2 As shown in Figure A, the cognitive index of mice in the model group was significantly lower than that in the blank control group (p<0.01), indicating that their cognitive function was impaired. In contrast, the cognitive index of mice in the MP drug treatment group was significantly higher than that in the aging model group, but there was no significant difference between them and the control group.

[0032] The water maze test results show that, Figure 2 As shown in B, during the navigation training, starting from day 2, the latency period of the model group mice was significantly longer than that of the blank control group, while the latency period of the MP drug treatment group mice was significantly shorter than that of the model group from day 3 onwards; Figure 2 As shown in Figure C, in the space exploration experiment, the number of times the MP drug-treated group mice crossed the platform was greater than that of the model group, but there was no statistically significant difference; furthermore, as... Figure 2 As shown in Figure D, there was no statistically significant difference in the average swimming speed among the three groups of mice, excluding the influence of exercise capacity on the results; indicating that MP treatment can improve age-related learning and memory deficits.

[0033] Example 3: Effects of MP on D-Gal-induced hippocampal LTP in aging mice I. Experimental Materials and Methods Twenty-four hours after the last administration of the drug in Example 2, the mice were anesthetized and euthanized, and the hippocampus tissue was quickly removed and placed in pre-cooled (0-4°C) artificial cerebrospinal fluid (ACSF) circulated with 95% O2 and 5% CO2. Coronal hippocampal slices with a thickness of 300 μm were prepared using a vibratory slicer. The slices were then incubated in continuously oxygenated ACSF at 32-34°C for at least 1 hour to restore activity. The slices were fixed in a perfusion tank, and the stimulating electrodes were placed in the Schaffer collateral pathway from CA3 to CA1. The recording glass microelectrode was placed in the radiative layer of the pyramidal cell layer in CA1. The stimulation intensity that could induce a 40% maximum fEPSP slope was determined by input-output (I / O) curves and used as the baseline stimulation intensity. After recording a stable baseline fEPSP for 10 minutes, high-frequency stimulation (4 x 100 Hz) was applied to induce LTP. EPSP was continuously recorded for at least 60 minutes after induction, and the slope change rate at each time point was calculated with the baseline fEPSP slope as 100%.

[0034] II. Experimental Results This embodiment investigated the effect of MP on D-Gal-induced long-term potentiation (LTP) in the hippocampus of aging mice at the animal level. The results are as follows: Figure 3 As shown, after the application of high-frequency stimulation, the fEPSP slope of mice in each group immediately increased. Among them, the fEPSP slope of mice in the blank control group increased significantly and remained at a high level, while the fEPSP slope of mice in the model group was significantly lower than that of the blank control group. After MP treatment, the fEPSP slope of mice was significantly higher than that of the model group throughout the recording period and approached the level of the blank control group. The above results indicate that the level of long-term enhancement in aging mice was significantly lower than that in the blank control group, and MP treatment can effectively reverse this damage and significantly increase the level of LTP.

[0035] In summary, this invention has demonstrated at the cellular and animal levels that methyl palmitate has significant anti-neuroaging effects and improves age-related cognitive impairment, and can be used to prepare drugs for the treatment and / or prevention of aging and related cognitive impairment.

[0036] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. The use of methyl palmitate in the preparation of drugs for the treatment and / or prevention of aging and related cognitive impairment.

2. The application according to claim 1, characterized in that, The molecular formula of the methyl palmitate is CH3(CH2). 14 CO2CH3, CAS number: 112-39-0, chemical structural formula: 。 3. The application according to claim 1, characterized in that, In this application, methyl palmitate is the sole active ingredient.

4. The application according to claim 1, characterized in that, The methyl palmitate inhibits the expression levels of aging-related proteins P53 and P16.

5. The application according to claim 1, characterized in that, The methyl palmitate can enhance the long-term potentiation of the hippocampus.

6. A drug for preventing aging and related cognitive impairment, characterized in that, The drug includes methyl palmitate and medically acceptable pharmaceutical excipients.

7. The drug for preventing aging and related cognitive impairment according to claim 6, characterized in that, Methyl palmitate is the sole active ingredient.

8. The use of methyl palmitate in the preparation of drugs for the treatment of neurodegenerative diseases.

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