Application of linoleamide in anti-aging and / or life extension
By adding linoleic amide to C. elegans and mouse embryonic fibroblasts, the effect of delaying aging and extending lifespan was studied, and the research problem of lack of effective anti-aging and prolonging lifespan in the prior art was solved, and the effect of significantly extending lifespan, improving physical mobility and inhibiting the expression of aging-related genes was achieved.
Patent Information
- Application Number
- CN202311143923.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-06
- Publication Date
- 2025-05-02
- Estimated Expiration
- 2043-09-06
AI Technical Summary
There is a lack of effective anti-aging and life-extending studies in the prior art, especially the application of linoleic amide in this area.
The effect of delaying aging and prolonging lifespan by adding linoleic amide to C. elegans and mouse embryonic fibroblasts was studied. Specifically, it includes adding linoleic amide to the culture medium and studying its effects on nematode lifespan, physical mobility, pharyngeal pump swallowing frequency, lipofuscin autofluorescence and cell aging-related gene expression through a variety of experimental methods.
Linoleic amide significantly extends the lifespan of C. elegans, improves its physical mobility and pharyngeal pump swallowing frequency, reduces lipofuscin autofluorescence, and inhibits the expression of aging-related genes, delaying the aging process of replicating senescent cell models.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to application of linoleamide in anti-aging and / or life extension, belonging to the technical field of anti-aging drugs. Background Art
[0002] Linoleamide, also known as 9,12-Octadecadienamide or linoleic acid amide, is a member of the primary fatty acid amide family of endogenous metabolites. Linoleamide was first discovered and identified in female luteal phase plasma in 1989. In 1995, linoleamide was isolated from the cerebrospinal fluid of sleep-deprived cats, rats, and humans. Related studies have shown that it can be used as an endogenous sleep inducer to induce sleep in cats, rats, and humans. Studies have shown that linoleamide can increase Ca2+ in the cytoplasm. + levels, which may be due to the effect of linoleamide on endoplasmic reticulum Ca2 + -ATPase inhibition. The researchers also found that linoleamide can inhibit the activity of phospholipase 2 (PLA2), thereby playing a certain protective role against diet-induced obesity and diabetes. The traditional Vietnamese medicinal ingredient, airplane grass, has always been widely used to treat diseases such as enteritis and burns for its anti-inflammatory and hemostatic effects. The researchers extracted the lipid component linoleamide from airplane grass, proving that it can inhibit lipopolysaccharide LPS-induced NF-kB activity and reduce NO production. In addition, the Chinese herbal medicine Epimedium has been proven to be a Chinese herbal medicine with anti-aging and lipid metabolism regulation, and epimedium is its main active ingredient. The study found that the level of linoleamide increased in the liver of elderly rats treated with epimedium, suggesting that it may be related to the relevant mechanism of action of epimedium, but whether linoleamide itself can directly regulate aging has not been reported, and there is no related research report on linoleamide in anti-aging and life extension. Summary of the invention
[0003] The purpose of the present invention is to provide the application of linoleamide in delaying aging and / or prolonging life, thereby expanding the application scope of linoleamide.
[0004] The present invention provides the use of linoleamide in preparing a product for delaying nematode aging and / or extending the life span of nematodes;
[0005] The animal is a mammal, specifically a human;
[0006] The animal is an invertebrate, specifically Caenorhabditis elegans.
[0007] The present invention also provides the use of linoleamide in the preparation of medicines and health products having any one of the following functions 1) to 5):
[0008] 1) Extend lifespan;
[0009] 2) Delay aging;
[0010] 3) Improve the survival rate;
[0011] 4) Improve motor function and health function;
[0012] 5) Inhibit lipofuscin deposition.
[0013] The present invention also provides a method for culturing Caenorhabditis elegans, comprising the steps of adding linoleamide to the culture medium;
[0014] The concentration of linoleamide is preferably 10 mM.
[0015] This invention uses Caenorhabditis elegans and mouse embryonic fibroblasts (MEF) as research objects, and studies the anti-aging effects of linoleamide from the aspects of nematode lifespan, body activity, pharyngeal pump swallowing frequency, lipofuscin autofluorescence and cell aging-related gene expression, providing a basis for the further development of linoleamide-related anti-aging drugs and health products.
[0016] The present invention discovers the application of linoleamide in prolonging life and delaying aging, provides a new use of linoleamide, expands the application scope of linoleamide, and has important economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 The result of linoleamide extending the lifespan of nematodes.
[0018] Figure 2 The result is that linoleamide improves the body activity of nematodes.
[0019] Figure 3 The result is that linoleamide increases the frequency of pharyngeal pump swallowing in nematodes.
[0020] Figure 4 This is the result of linoleamide reducing the autofluorescence of lipofuscin.
[0021] Figure 5 The result is that linoleamide delays the aging process in the replicative senescence cell model. DETAILED DESCRIPTION
[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0023] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.
[0024] Escherichia coli OP50 and wild-type Caenorhabditis elegans strain N2 were kindly provided by Zhang Hong's laboratory at the Institute of Biophysics, Chinese Academy of Sciences (Gangming Zhang et al., Cell 174, 1492-1506, September 6, 2018).
[0025] Linoleamide was purchased from Shanghai MacLean Biochemical Technology Co., Ltd.
[0026] Mouse embryonic fibroblasts (MEFs) were isolated from mouse embryos at E11.5-E13.5.
[0027] Example 1: Linoleamide prolongs lifespan of nematodes
[0028] The culture of nematodes was carried out according to the method used by Brenner. L4 nematodes were selected on NGM dishes containing E. coli OP50, and the dishes were placed in a biochemical incubator at 20°C for culture. The dishes were turned every 3 days. The synchronization of nematodes was carried out by sodium hypochlorite lysis method. The nematodes in the egg-laying stage were washed off the NGM dishes with sterilized M9 solution, centrifuged at 3000rpm for 3min, and this step was repeated 3 times. 5.5mL lysis solution (250μL NaClO, 250μl 10MNaOH and 5mL pressurized water were added to the tube, and the mixture was prepared immediately before use and kept away from light) and vortexed for 10min until all the worm bodies disappeared. 5ml M9 solution was added to the tube to stop the lysis, and the tube was centrifuged at 3000rpm for 3min. The supernatant was discarded and the precipitate (the egg precipitate was white) was collected. The precipitate was washed repeatedly with M9 solution for 3 times, and then transferred to M9 solution for culture at 20°C. After 20 hours, the L1 nematodes were transferred to NGM plates containing E. coli OP50. The nematodes developed to the L4 stage in about 48 hours, completing synchronization.
[0029] Linoleamide was fed to nematodes by mixing with Escherichia coli OP50. The nematode lifespan experiment was divided into a control group and a linoleamide-added group. The concentration of linoleamide in the OP50 bacterial solution was two doses: 200μM and 10mM. The control group was replaced with an equal volume of DMSO. 120 L4 nematodes were selected in parallel for each group and transferred to NGM plates coated with the corresponding OP50 bacterial solution and cultured at 20°C. This was recorded as the 0th day of the nematode lifespan. In the early stage of the nematode life (0-10 days), NGM plates containing 5μM FUDR were used to inhibit nematode egg laying. On the 11th day, NGM plates without FUDR were used. In order to ensure the concentration of the drug, sufficient food and prevent contamination, the nematodes were transferred to a new culture dish every 3 days. The number of nematodes that survived, died and lost was recorded every day until all nematodes died. The nematodes were judged dead when they were touched lightly with platinum wire and there was no movement or swallowing of the worm body. Nematodes that burrow into the culture medium, are lost, crawl onto the wall of the culture dish and die, have their genitals burst, or form worm bags are not counted.
[0030] The experimental data were statistically analyzed using GraphPad Prism 8 software to analyze the overall survival rate and average lifespan of the nematodes. When the overall lifespan and average lifespan of the nematodes in the experimental group were higher than those in the control group, and P < 0.05, it was considered that linoleamide could prolong the lifespan of the nematodes.
[0031] The experimental results are as follows Figure 1 As shown, it can be seen that linoleamide can extend the lifespan of Caenorhabditis elegans, and the effect of extending lifespan is most obvious when the concentration is 10mM.
[0032] Example 2: Linoleamide increases the activity of nematodes
[0033] The experiment was divided into a control group and a 10 mM linoleamide group (experimental group). The control group was a DMSO group, and the experimental group was a 10 mM linoleamide group.
[0034] In the process of measuring the life span of C. elegans, the number of body bends of the experimental and control groups was measured on days 3, 7, 11, 15, 19, and 23. A nematode was placed on an NGM dish without OP50 and allowed to move freely for about 1 minute. The number of body bends within 30 seconds was recorded. One bend was defined as the movement of one wavelength of the nematode relative to the long axis of the body. The number of nematodes measured in each group of experiments was no less than 15.
[0035] The experimental data obtained in this example were statistically analyzed using GraphPad Prism 8 software using two-way anova, with p<0.05 considered statistically significant.
[0036] The experimental results are as follows Figure 2 As shown, it can be seen that linoleamide can increase the body activity of Caenorhabditis elegans.
[0037] Example 3: Linoleamide increases the frequency of pharyngeal pump swallowing in nematodes
[0038] The experiment was divided into a control group and a 10 mM linoleamide group (experimental group). The control group was a DMSO group, and the experimental group was a 10 mM linoleamide group.
[0039] During the determination of the life span of C. elegans, the pharyngeal pumping frequency of the experimental and control groups was measured on days 3, 7, 11, 15, and 19. The pharyngeal pumping rate of the nematodes within 30 seconds was recorded directly on the experimental dish using a microscope containing a CCD image sensor. The number of nematodes measured in each group of experiments was no less than 15.
[0040] The experimental data obtained in this example were statistically analyzed using GraphPad Prism 8 software using two-way anova, with p<0.05 considered statistically significant.
[0041] The experimental results are as follows Figure 3 As shown, it can be seen that linoleamide can increase the swallowing frequency of pharyngeal pump in Caenorhabditis elegans.
[0042] Example 4: Linoleamide reduces nematode lipofuscin autofluorescence
[0043] The experiment was divided into a control group and a 10 mM linoleamide group (experimental group). The control group was a DMSO group, and the experimental group was a 10 mM linoleamide group.
[0044] In the process of measuring the life span of C. elegans, the fluorescence level of lipofuscin in the body of the experimental group and the control group was measured at 4, 8, 12, 16, 20 and 24 days. The experimental steps are as follows: Weigh 2g agarose and dissolve it in 100mL 1×TAE buffer (2% agar solution), stir and mix well, and heat it in a microwave for 3 minutes to make it completely melted. Prepare a slide, drop a drop of agar solution in the center of the slide, cover it with a cover glass while it is hot, and gently flatten the agar with your fingers to avoid the formation of bubbles. After the agar is completely solidified, remove the cover glass with tweezers and drop 20μL M9 buffer in the center of the agar. Anesthetize the nematodes with 5mM levamisole. After the nematodes have no reaction to the platinum wire, pick the nematodes into the M9 solution in the center of the agar and cover them with a cover glass. Photograph the fluorescence of lipofuscin in the nematode intestine under a 10x microscope, with an excitation wavelength of 340-380nm and an emission wavelength of 430nm. Image J software was used to analyze the fluorescence intensity of lipofuscin.
[0045] The experimental data obtained in this example were statistically analyzed using GraphPad Prism 8 software using two-way anova, with p<0.05 considered statistically significant.
[0046] The experimental results are as follows Figure 4 As shown, it can be seen that linoleamide can reduce the autofluorescence of lipofuscin in Caenorhabditis elegans.
[0047] Example 5: Linoleamide delays the aging process of replicative senescence cell models and inhibits the expression of aging-related genes
[0048] MEF culture medium is prepared by DMEM basal medium, 15% FBS and penicillin-streptomycin. C57 / 6J pregnant mice of 11.5-13.5 days of pregnancy were obtained, killed and soaked in 75% ethanol for 15 minutes. The embryonic head, limbs, tail and internal organs were removed in a clean bench, and the remaining parts were placed in an EP tube containing 200μl PBS. The tissue was cut into pieces with scissors, 1ml pancreatic enzyme was added and blown evenly, and the cells were placed in a 37℃ incubator for digestion for 10 minutes, and the digestion was terminated with an equal volume of culture medium. Centrifuge at 800r / min for 3min, discard the supernatant, resuspend the cell pellet with culture medium, plant one embryo in a 10cm dish, change the medium after 6h of attachment, and pass the culture medium at a ratio of 1:2 every 3d. P3 control group cells, P6 control group cells and P6 experimental group cells were fully lysed with TRIzol, and after extracting total cell RNA, the RNA was reverse transcribed into cDNA using PrimeScriptTMRT Master Mix, and the expression of aging-related genes was detected by qPCR using PowerUpTM SYBR Green premix.
[0049] The experimental data obtained in this example were statistically analyzed using GraphPad Prism 8 software, and the two groups were compared using a paired t-test, with p < 0.05 considered statistically significant.
[0050] The experimental results are as follows Figure 5 As shown, it can be seen that linoleamide reduces the expression of senescence-related genes in the replicative senescence cell model.
Claims
1. Application of linoleamide in the preparation of drugs for delaying animal aging and / or prolonging animal lifespan.
2. The use according to claim 1, characterized in that: The animal is a mammal.
3. The use according to claim 2, characterized in that: The mammal is a human.
4. The use according to claim 1, characterized in that: The animal is an invertebrate.
5. The use according to claim 4, characterized in that: The invertebrate is Caenorhabditis elegans.
6. The use according to any one of claims 1 to 5, characterized in that: The application is any one of the functions shown in 1)-5) below: 1) Extend lifespan; 2) Delaying aging; 3) Improve the survival rate; 4) Improve motor function and health function; 5) Inhibit lipofuscin deposition.