Method for evaluating the effects of different trace components in wine on cellular autophagy levels

Abstract

The present application is used in the field of food and discloses a method for evaluating the degree of influence of trace components in wine on the level of cellular autophagy, comprising the following steps: Step 1, preparing a single cell suspension of human hepatocytes (LO2) stably expressing GFP-RFP-LC3 protein; Step 2, adding 1.5×10 4 The LO2 cells with a cell / mL were inoculated into a 6-well plate and covered with a glass for 24 hours until they adhered to the wall; Step three, 2 mL of wine containing 80 μL of pure alcohol or wine samples containing different trace components of wine were added to the cells and cultured at 37°C and 5% CO2 for 24 hours. Step four, fix in 4% paraformaldehyde for 15 minutes. Step five, wash the slides with PBS and dry them, then add an anti-fading agent containing DAPI, and place the side covered with cells on the anti-fading agent. Step six, observe the red and green fluorescence using a laser confocal microscope, and judge the effect of the trace components of the wine on the level of cellular autophagy based on the different fluorescence. The present application can quickly evaluate the degree of influence of the trace components in the wine on the level of cellular autophagy by observing the different fluorescence as mentioned above.

Description

Field of the invention:

[0001] The present invention relates to the field of food, and in particular to a method for evaluating the degree of influence of different trace components in wine on the level of cellular autophagy. Background technology:

[0002] Compared to other distilled spirits, baijiu, in addition to its primary ingredient, ethanol, contains a richer variety of flavor compounds, including over 2,000 trace compounds. These compounds account for 2%-3% of the total weight, and include a variety of functional components, including polyphenols, terpenes, pyrazines, peptides, and other active ingredients. Ethanol lacks the ability to alleviate heart disease; this function may be more influenced by other trace components. For example, resveratrol has antioxidant properties, prevents cardiovascular disease, and prolongs the lifespan of human organs; 4-ethylguaiacol can regulate pro-inflammatory signaling pathways and reduce inflammatory cytokines; phylloxetine and β-caryophyllene can increase the concentration of the non-enzymatic antioxidant GSH in the body, while also enhancing the antioxidant capacity of cells; and lipopeptides have certain anti-cancer and antiviral properties.

[0003] Autophagy is a fundamental cellular metabolic process that promotes cellular homeostasis, differentiation, development, and survival. It maintains cell survival by degrading molecules and subcellular components, including nucleic acids, proteins, lipids, and organelles, through lysosomes. Autophagy is a highly selective cellular clearance pathway involved in maintaining cellular tissue homeostasis and is closely linked to health, particularly aging and disease. Impaired autophagy is considered a hallmark of aging. Studies in long-lived mutant animals have shown that delayed aging is associated with increased autophagy, and genetic or pharmacological approaches to upregulate autophagy can extend animal lifespan. Excessive alcohol intake can cause alcoholic liver damage and even hepatocellular carcinoma, further damaging proteins in the body and leading to the negative effects of disease and aging. Impaired autophagy in lipid metabolism is the pathological basis for lipid peroxidation and cell damage. Alcoholic fatty liver disease (AFLD) results from excessive alcohol consumption, leading to oxidative stress in the liver, excessive lipid droplet accumulation in hepatocytes (steatosis), mitochondrial damage, and cell death. Autophagy acts as a defense mechanism to prevent lipid peroxidation, thereby protecting hepatocytes. Chinese liquor may mitigate damage to hepatocytes by affecting autophagic activity.

[0004] Currently, there are no unified standards for healthy drinking, and no established experimental procedures. Animal experiments assessing healthy drinking not only need to consider individual differences, but also face high experimental costs and lengthy experimental processes, hindering companies and consumers from quickly and conveniently evaluating the health of alcohol and its ingredients. Therefore, this application provides a method for effectively and rapidly evaluating the impact of different trace components in alcoholic beverages on cellular autophagy levels, enabling rapid and convenient advancement of healthy beverage design and catering to the needs of healthy drinking consumers. Summary of the invention:

[0005] To address these needs, the inventors prepared normal human hepatocytes that stably express the GFP-RFP-LC3 protein and treated these cells with different wine samples containing different trace components found in wine. Laser confocal microscopy was used to observe cell morphology and analyze the effects on autophagy in normal human hepatocytes expressing the GFP-RFP-LC3 protein. This allows for a rapid, simple, and effective preliminary assessment of the health impact of different trace components in wine.

[0006] In one aspect, the present application provides a method for evaluating the effects of different trace components in wine on the level of cellular autophagy, characterized in that the method comprises the following steps:

[0007] Step 1: Human hepatocytes L02 in the logarithmic growth phase that can stably express GFP-RFP-LC3 protein are digested with trypsin to prepare a single-cell suspension;

[0008] Step 2: Place three cover glasses evenly in each well of a 6-well plate. First, add a small amount of culture medium to the wells of the 6-well plate (to ensure close contact between the glass slide and the culture dish), and make the glass slide fit tightly. 4 LO2 cells stably expressing GFP-RFP-LC3 were seeded into 6-well plates at a density of 1 mL per well. The plates were covered and incubated at 37°C, 5% CO2 for 24 hours until the cells adhered.

[0009] Step 3: Remove the cell culture medium, wash twice with PBS, and then add 2 mL of cell culture mixture containing 80 μL of sample to the cell wall. Incubate at 37°C, 5% CO2 for 24 hours.

[0010] Step 4: Remove the supernatant, wash twice with PBS, and then fix in 4% paraformaldehyde for 15 minutes;

[0011] Step 5: Wash twice with PBS, pick up the slide with tweezers, place the slide on filter paper to dry, and add antiquenching agent containing DAPI on the slide, and place the side covered with cells on the antiquenching agent;

[0012] Step 6. Observe fluorescence using a laser confocal microscope.

[0013] Furthermore, the amino acid sequence of the GFP-RFP-LC3 protein is SEQ ID NO.1.

[0014] Furthermore, the preparation method of human liver cells L02 capable of stably expressing GFP-RFP-LC3 protein includes: cloning or synthesizing GFP, RFP, and LC3 genes, assembling a GFP-RFP-LC3 plasmid; co-transfecting the prepared GFP-RFP-LC3 plasmid with pMD2G and PSApX2 packaging plasmids into 293T cells to prepare a lentivirus carrying the GFP-RFP-LC3 gene; and using the packaged lentivirus to infect liver tissue cells.

[0015] Furthermore, the concentration range of the trace components in the wine is 0ug / mL to 400ug / mL.

[0016] Furthermore, the trace components of the wine include but are not limited to lactic acid, acetic acid, propionic acid, formic acid, isobutyric acid, n-butyric acid, pyruvic acid, isovaleric acid, 2-ethylbutyric acid, valeric acid, chloride ion, hexanoic acid, nitrate, malic acid, tartaric acid, sulfate, oxalic acid, fumaric acid, phosphoric acid, citric acid, n-propanol, n-butanol, isobutanol, sec-butanol, amyl alcohol, isopentanol, β-phenylethanol, glycerol, 2,3-butanediol, erythritol, mannitol, arabitol, acetaldehyde, acetal, 1,1-diethoxyisobutane, 1,1-diethoxyisopentane, propionaldehyde, isobutyraldehyde, isovaleraldehyde, furfural, diacetyl, hexanone, 2, 3-Butanedione, 3-hydroxybutanone, ethyl acetate, ethyl hexanoate, ethyl lactate, ethyl butyrate, ethyl formate, isoamyl acetate, ethyl isovalerate, ethyl propionate, ethyl valerate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, diethyl succinate, ethyl laurate, ethyl phenylacetate, ethyl palmitate, ethyl oleate, ethyl linoleate, benzaldehyde, β-phenylethanol, ethyl benzoate, ethyl phenylacetate, phenol, guaiacol, benzoic acid, vanillin, furoic acid, tetramethylpyrazine, trimethylpyrazine, 2,6-dimethylpyrazine, glutamic acid, alanine, aspartic acid, tyrosine, arginine, glycine.

[0017] Furthermore, the sample may be an edible alcohol standard solution and / or a wine sample.

[0018] Furthermore, the wine samples are all 50% alc / vol.

[0019] Furthermore, the preparation method of the edible alcohol standard solution is to remove all trace components in the Russian vodka by distillation and activated carbon, retaining only ethanol and water, and then preparing solutions of various alcohol concentrations with ultrapure water.

[0020] Furthermore, fluorescence was observed using a laser confocal microscope. After microscopic imaging, yellow spots formed by the mixture of red and green fluorescence were autophagosomes, and a decrease in green fluorescence indicated an increase in the level of autophagic flux.

[0021] On the other hand, the present application provides the application of the above-mentioned method for rapidly evaluating the degree of influence of different trace components in wine on the level of cellular autophagy to evaluate the health effects of alcoholic beverages. Description of the drawings:

[0022] Figure 1 This is a schematic diagram of the LC3 red and green fluorescent protein expression results under a laser confocal microscope after the cells were treated with nutrient conditions for 2 hours;

[0023] Figure 2 Schematic diagram of LC3 red and green fluorescent protein expression under laser confocal microscopy after cells were treated with edible alcohol and wine containing 50 μg / mL eugenol for 24 hours;

[0024] Figure 3 This is a schematic diagram of the LC3 red and green fluorescent protein expression results under a laser confocal microscope after cells were treated with edible alcohol, wine sample 1# and wine sample 2# for 24 hours. Specific implementation method:

[0025] The following is further described in detail through specific implementation methods:

[0026] Human hepatocytes LO2 stably expressing GFP-RFP-LC3 protein were purchased from XX or prepared by XX; the sequence of the GFP-RFP-LC3 protein expressed therein is shown in SEQ ID NO.1.

[0027] Example 1

[0028] Step 1: LO2 cells and 293T cells were purchased from the National Laboratory Cell Resource Sharing Platform.

[0029] Step 2: Complete the plasmid structure design based on the pCDH plasmid; extract hepatocyte cellular RNA and prepare a cDNA library; search the LC3 sequence number in NCBI, Gene ID: 66734, and prepare the LC3 gene from the cDNA library; prepare the EGFP gene based on Clontech's EGFP-C1 plasmid; prepare the mRFP gene based on Addgene's pcDNA3-mRFP plasmid; after multiple assembly steps, complete the preparation of the pCDH-GFP-RFP-LC3-puro plasmid.

[0030] Step 3: One day before transfection, seed an appropriate amount of 293T cells in a culture dish until the cell density reaches 70-90% at the time of transfection.

[0031] Step 4: Take 20 μg of plasmid (the virus is a 3-plasmid system, pMD2G:PSApX2:target plasmid = 4:6:10 ug) and add it to 400 μl of physiological saline, and mix well by pipetting.

[0032] Step 5: Take 8 μl of Vigofect transfection reagent, add it to 400 μl of normal saline, mix gently, and let it stand at room temperature for 5 minutes.

[0033] Step 6. Add the plasmid diluted in step 4 to the transfection reagent diluted in step 5, pipette gently to mix, and let stand at room temperature for 15 minutes.

[0034] Step 7: Add the transfection solution dropwise to the cell culture medium, gently mix the medium, and place it in the cell culture incubator. After 4-6 hours, replace the medium with fresh medium. Observe the cells 24-48 hours after transfection, replace the medium with fresh medium after 24 hours, and collect the viral supernatant after 48-72 hours.

[0035] Step 8: One day before transfection, LO2 cells were seeded into a 6-well plate at 60% confluency per well.

[0036] Step 9: Treat cells with 1 ml of the viral supernatant from Step 9 per well and incubate for 36 hours. Add puromycin (2 μL, 1-1.5 μg / mL) and incubate for 24 hours. Observe the cell status and proceed with cell passage and single cell sorting.

[0037] Step 10: Sort the cells by laser confocal microscopy and determine the degree of fluorescence.

[0038] Example 2

[0039] Step 1: Human hepatocytes LO2 in the logarithmic growth phase that can stably express GFP-RFP-LC3 protein are digested with trypsin to prepare a single-cell suspension.

[0040] Step 2: Place three cover glasses evenly in each well of a 6-well plate. First, add a small amount of culture medium to the wells of the 6-well plate (to ensure close contact between the glass slide and the culture dish), and make the glass slide fit tightly. 4 LO2 cells stably expressing GFP-RFP-LC3 were seeded into 6-well plates at a density of 1 mL per well. The plates were covered and incubated at 37°C, 5% CO2 for 24 hours until the cells adhered.

[0041] Step 3: Remove the cell culture medium and wash twice with PBS. After the cells adhere, add 2 mL of cell culture mixture. The cell culture mixture for each group consists of: 80 μL of a 50% alc / vol standard ethanol solution; 80 μL of a 50% alc / vol standard ethanol solution containing 50 μg / mL eugenol. Incubate at 37°C, 5% CO2 for 24 hours.

[0042] Step 4: Remove the supernatant, wash twice with PBS, and then fix in 4% paraformaldehyde for 15 minutes.

[0043] Step 5: Wash twice with PBS, pick up the slide with tweezers, place the slide on filter paper to dry, add anti-quenching agent containing DAPI on the slide, and place the side covered with cells on the anti-quenching agent.

[0044] Step 6. Observe fluorescence using a fluorescence confocal microscope.

[0045] Results: Laser confocal microscopy revealed that after 24 hours of treatment with eugenol at a final concentration of 50 μg / mL, a significant number of autophagosomes formed within the cells, and LC3 protein was recruited to the autophagosome surface. The presence of slightly more red LC3 protein than green LC3 protein indicated that autophagy was induced and that the autophagosomes matured and fused with lysosomes to form autolysosomes. While alcohol had no significant effect on autophagy, eugenol, a component of alcohol, was able to induce the initiation, development, and maturation of autophagy.

[0046] Example 3

[0047] Step 1: Human hepatocytes LO2 in the logarithmic growth phase that can stably express GFP-RFP-LC3 protein are digested with trypsin to prepare a single-cell suspension.

[0048] Step 2: Place three cover glasses evenly in each well of a 6-well plate. First, add a small amount of culture medium to the wells of the 6-well plate (to ensure close contact between the glass slide and the culture dish), and make the glass slide fit tightly. 4 LO2 cells stably expressing GFP-RFP-LC3 were seeded into 6-well plates at a density of 1 mL per well. The plates were covered and incubated at 37°C, 5% CO2 for 24 hours until the cells adhered.

[0049] Step 3: Remove the cell culture medium and wash twice with PBS. After the cells adhere to the wall, add 2 mL of cell culture mixture. The cell culture mixture for each group is as follows: 80 μL of 50% alc / vol standard edible alcohol solution, 80 μL of alcohol sample 1, and 80 μL of alcohol sample 2. Incubate at 37°C, 5% CO2 for 24 hours.

[0050] Step 4: Remove the supernatant, wash twice with PBS, and then fix in 4% paraformaldehyde for 15 minutes.

[0051] Step 5: Wash twice with PBS, pick up the slide with tweezers, place the slide on filter paper to dry, add anti-quenching agent containing DAPI on the slide, and place the side covered with cells on the anti-quenching agent.

[0052] Step 6. Observe fluorescence using a fluorescence confocal microscope.

[0053] The results showed that alcohol consumption had no significant effect on cellular autophagy, and that different alcohol samples had varying effects on autophagy. After 24 hours of treatment with alcohol sample 1, a certain number of autophagosomes formed within the cells, and LC3 protein was recruited to the autophagosome surface. The presence of slightly more red LC3 protein than green LC3 protein indicated that autophagy was induced and that the autophagosomes matured and fused with lysosomes to form autolysosomes. In contrast, alcohol sample 2 showed no significant changes in red and green fluorescence, suggesting that it had no significant effect on autophagy.

Claims

1. A method for evaluating the influence of different trace components in wine on the level of cellular autophagy, characterized in that: The following steps are involved: Step 1: Human hepatocytes L02 stably expressing GFP-RFP-LC3 protein in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension; Step 2: Place 3 coverslips evenly in each well of a 6-well plate. Add a small amount of culture medium to the wells of the 6-well plate to make the coverslips fit tightly. 4 L02 cells stably expressing GFP-RFP-LC3 were seeded into a 6-well plate at a density of 1 mL per well. The 6-well plate was covered and cultured at 37°C, 5% CO2 for 24 h until the cells attached to the wall. Step 3: Remove the culture medium, wash twice with PBS, and then add 2 mL of cell culture mixture containing 80 μL of sample to the cell wall. Incubate at 37°C, 5% CO2 for 24 hours. Step 4: Remove the supernatant, wash twice with PBS, and then fix in 4% paraformaldehyde for 15 minutes; Step 5: Wash twice with PBS, pick up the slide with tweezers, place the slide on filter paper to dry, and add antiquenching agent containing DAPI on the slide, and place the side covered with cells on the antiquenching agent; Step 6: Observe fluorescence using a laser confocal microscope; The amino acid sequence of the GFP-RFP-LC3 protein is SEQ ID NO.

1. The preparation method of human hepatocytes L02 stably expressing the GFP-RFP-LC3 protein comprises: cloning or synthesizing GFP, RFP, and LC3 genes, and assembling a GFP-RFP-LC3 plasmid; The GFP-RFP-LC3 plasmid was co-transfected with the pMD2G and PSApX2 packaging plasmids into 293T cells to prepare lentivirus carrying the GFP-RFP-LC3 gene; the lentivirus was used to infect liver tissue cells.

2. The method according to claim 1, wherein the concentration range of the different trace components in the wine is 0 ug / mL to 400 ug / mL.

3. The method according to claim 1, wherein the different trace components in the wine are selected from lactic acid, acetic acid, propionic acid, formic acid, isobutyric acid, n-butyric acid, pyruvic acid, isovaleric acid, 2-ethylbutyric acid, valeric acid, chloride ion, hexanoic acid, nitrate, malic acid, tartaric acid, sulfate, oxalic acid, fumaric acid, phosphoric acid, citric acid, n-propanol, n-butanol, isobutanol, sec-butanol, amyl alcohol, isopentanol, glycerol, 2,3-butanediol, erythritol, mannitol, arabitol, acetaldehyde, acetal, 1,1-diethoxyisobutane, 1,1-diethoxyisopentane, propionaldehyde, isobutyraldehyde, isovaleraldehyde, furfural, diacetyl, One or more of hexanone, 3-hydroxybutanone, ethyl acetate, ethyl hexanoate, ethyl lactate, ethyl butyrate, ethyl formate, isoamyl acetate, ethyl isovalerate, ethyl propionate, ethyl valerate, ethyl heptanoate, ethyl octanoate, ethyl nonanoate, ethyl decanoate, diethyl succinate, ethyl laurate, ethyl palmitate, ethyl oleate, ethyl linoleate, benzaldehyde, β-phenylethanol, ethyl benzoate, ethyl phenylacetate, phenol, guaiacol, benzoic acid, vanillin, furoic acid, tetramethylpyrazine, trimethylpyrazine, 2,6-dimethylpyrazine, glutamic acid, alanine, aspartic acid, tyrosine, arginine, and glycine. The method according to claim 1 , wherein the sample is a wine sample.

5. The method of claim 4, wherein the wine sample is 50% alc / vol.

6. The method according to claim 1, wherein fluorescence is observed using a laser confocal microscope. After microscopic imaging, yellow spots formed by the mixture of red and green fluorescence are autophagosomes, and a decrease in green fluorescence indicates an increase in the level of autophagic flux.

7. Use of the method according to any one of claims 1 to 6 in evaluating the health effects of alcoholic beverages.

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