CN112451673A - Application of mitochondrial function intervention material in preparation of health-care product or medicine for improving mitochondrial function and screening method thereof - Google Patents

Application of mitochondrial function intervention material in preparation of health-care product or medicine for improving mitochondrial function and screening method thereof Download PDF

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CN112451673A
CN112451673A CN202011433402.2A CN202011433402A CN112451673A CN 112451673 A CN112451673 A CN 112451673A CN 202011433402 A CN202011433402 A CN 202011433402A CN 112451673 A CN112451673 A CN 112451673A
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mitochondrial
cell
mitochondrial function
palmitic acid
mitochondria
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李言
王立
聂陈志鹏
贺瑞坤
张旭光
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BY Health Co Ltd
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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5041—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects involving analysis of members of signalling pathways
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria
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    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Abstract

The invention discloses an application of a mitochondrial function intervention material in preparing health care products or medicines for improving mitochondrial functions and a screening method, wherein a mitochondrial damage model is constructed by adopting palmitic acid; determining acylcarnitine by an LC-MS/MS method; detecting the respiratory function by using a Seahorse extracellular flux analyzer; detecting the content of mitochondrial DNA by quantitative PCR; real-time quantitative PCR detection of mRNA level expression of genes mainly related to mitochondria; detecting related genes and oxidative phosphorylation complex proteins of mitochondria in skeletal muscle by using a Western blot method; detecting the MDA content and the NAD +/NADH content by using a kit; and observing the ultrastructure of cell mitochondria by adopting a transmission electron microscope and carrying out morphological analysis. Compared with the existing evaluation screening system, the evaluation screening system provided by the invention is more scientific, accurate and efficient.

Description

Application of mitochondrial function intervention material in preparation of health-care product or medicine for improving mitochondrial function and screening method thereof
Technical Field
The invention belongs to the field of health care products or medicines, and particularly relates to application of a mitochondrial function intervention material in preparation of health care products or medicines for improving mitochondrial functions and a screening method thereof.
Background
Mitochondria are an important component of eukaryotic cells, and have been considered as intracellular energy factories responsible for providing the cells with the necessary energy to perform their daily functions. The energy required by human cells is mainly provided by mitochondria, and it is worth noting that mitochondria function is getting worse with age. However, more and more research has found that mitochondria function more than "cellular energy sites", and that they are involved in the regulation of various cellular functions, has a profound link to many human diseases, including cell signaling, metabolism, autophagy, aging, and tumorigenesis, all of which are related to the quality and activity of mitochondria. Therefore, increasing researchers are concerned about how to improve the functions and health of mitochondria.
In addition, although there are a large number of nutritional supplements on the market that are declared to be targeted to mitochondria, most of them have problems of unclear specific efficacy, excessively exaggerated efficacy, unclear mechanism of action, and the like. At present, related technologies for mitochondrial function detection are various in types and different in means, and a relatively universal method comprises a related gene protein detection technology, a Seahorse mitochondrial metabolic capability detection technology, a transmission scanning electron microscope observation technology and a related metabolite identification technology. However, the function measurement of the mitochondria-related nutritional supplement has the problems of non-uniform detection indexes, lagged detection technology, single detection means, incomplete detection and the like. These problems cause the phenomena of uneven quality of related products and mixed real and fake products, such as fish and dragon, on the market.
Disclosure of Invention
In order to solve the technical problems, the invention mainly aims to provide an application of a mitochondrial function intervention material in preparing health care products or medicines for improving mitochondrial functions.
The invention also aims to provide a screening method of the mitochondrial function intervention material, which is more scientific, accurate and efficient compared with the existing evaluation screening system.
The invention is realized by the following technical scheme:
the invention provides application of a mitochondrial function intervention material in preparation of health care products or medicines for improving mitochondrial functions, wherein the mitochondrial function intervention material is one or a mixture of more of Nicotinamide Mononucleotide (NMN), holothurian peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or centella asiatica extract, and preferably NMN or holothurian peptide.
According to the invention, Palmitic Acid (PA) is adopted to construct a mitochondrial damage model, and researches show that NMN, sea cucumber peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or asiatic pennywort herb extract can obviously reverse the accumulation of acyl carnitine caused by PA, especially the content of medium-long chain acyl carnitine, and the basic oxygen consumption, ATP generation and maximum oxygen consumption are obviously increased. PCR results show that most of mitochondrion-related genes, particularly PPARD, Sirt3, OPA1, HSP60 and CPT1b, in NMN and sea cucumber peptide-treated C2C12 cells are significantly changed, and mRNA expression is increased hundreds of times. NMN and Stichopus japonicus peptide can also increase Cytc-1 protein level in C2C12 cells, and respiratory chain complex I also tends to increase. The NMN and the sea cucumber peptide can reduce excessive ROS and MDA overexpression caused by PA, activate SOD expression, and meanwhile, the NMN can obviously increase the ratio of NAD +/NAD. The mitochondrial morphology analysis image shows that PA treatment can cause partial mitochondria to swell, the volume of the mitochondria becomes large, and the internal ridge structure is damaged, so that the mitochondrial structure is disordered; NMN and sea cucumber peptide treatment can reverse damage of PA to mitochondria, so that the mitochondria volume is reduced, the mitochondria number is increased, the ridge structure is recovered to be normal and clearly visible, and the mitochondria morphology tends to be normal.
The research shows that NMN, sea cucumber peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or asiatic pennywort herb extract can obviously improve mitochondrial function. The invention adds effective dose of NMN, sea cucumber peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or asiatic centella extract as active ingredients into carriers acceptable in pharmacy or health-care food science, and prepares medicaments, foods or nutritional supplements and the like according to the conventional method in the field for treating or preventing diseases related to mitochondrial function.
The invention also provides a screening method of the mitochondrial function intervention material, which comprises the following steps:
(1) constructing a mitochondrial damage model by using palmitic acid;
(2) determining acylcarnitine by an LC-MS/MS method;
(3) detecting the respiratory function by using a Seahorse extracellular flux analyzer;
(4) detecting the content of mitochondrial DNA by quantitative PCR;
(5) real-time quantitative PCR detection of mRNA level expression of genes mainly related to mitochondria;
(6) detecting related genes and oxidative phosphorylation complex proteins of mitochondria in skeletal muscle by using a Western blot method;
(7) detecting the MDA content and the NAD +/NADH content by using a kit;
(8) and observing the ultrastructure of cell mitochondria by adopting a transmission electron microscope and carrying out morphological analysis.
According to the invention, a mitochondrial damage model is constructed by adopting Palmitic Acid (PA), a real-time metabolic analysis technology of Seahorse mitochondria and an acyl carnitine detection technology are adopted as main bodies, and a set of complete preliminary screening and function evaluation systems of mitochondrial function intervention materials are established by combining an immunoblotting test, a real-time quantitative PCR experiment, a kit detection and an electron microscope observation technology, so that the materials with the mitochondrial function intervention function are screened out.
As a further preferred technical solution of the present invention, the step (1) of constructing the mitochondrial damage model by using palmitic acid specifically comprises the following steps:
a. cell culture
Selecting C2C12 myoblasts, and putting the myoblasts into a growth culture medium for culture;
b. cell passage
Sucking out the growth medium when the cell fusion rate reaches 80-85%, washing the cells with PBS, adding pancreatin to digest the cells, and adding trypsin when the cell fusion rate reaches 80-85%
When 50-70% of cells become bright and round, absorbing pancreatin, adding a growth culture medium to stop pancreatin digestion, shedding digested cells to form a cell suspension, centrifuging, absorbing a supernatant, adding the growth culture medium, and transferring the cell suspension to a culture dish added with the growth culture medium according to a specific passage ratio for culture;
c. cell differentiation
Sucking out the growth medium when the cell growth fusion rate reaches 75-80%, and adding a differentiation medium for differentiation;
d. raw material treatment
Sucking up a differentiation culture medium in differentiated C2C12 myoblasts, rinsing with PBS, adding a palmitic acid solution and a raw material, culturing the cells added with the raw material in a cell culture box, sucking a cell culture solution, and storing at-80 ℃ for later use.
Preferably, the growth medium contains 10% FBS (fetal bovine serum) and 1% P/S (diabody, 100U/mL penicillin and 100. mu.g/mL streptomycin).
Preferably, the differentiation medium contains 2% HS (horse serum) and 1% P/S (diabody, 100U/mL penicillin and 100. mu.g/mL streptomycin).
Preferably, the cell culture conditions are 37 ℃ and 5% CO2。
Preferably, the preparation method of the palmitic acid solution comprises the following steps: weighing palmitic acid, and dissolving the palmitic acid in absolute ethyl alcohol to obtain a palmitic acid ethanol solution; weighing BSA and dissolving in PBS to obtain a PBS solution; the ethanol palmitic acid solution was added to the PBS solution to prepare a palmitic acid solution having a final concentration of 5 mM.
A large number of research results show that the development process of aging is closely related to mitochondrial dysfunction. In the aging process, the mitochondria biology changes, and the mitochondria generate free radicals and the damage of the free radicals to the mitochondria plays a role in promoting the development of aging; mitochondrial DNA mutations and mitochondrial regulation of the progression of cell death are closely related to the progression of senescence.
According to the screening method, NMN, sea cucumber peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or asiatic pennywort herb extract are obtained through research, mitochondrial functions can be obviously improved, and the screening method further has an anti-aging effect and can be applied to the fields of anti-aging food, health-care food, medicines and the like.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides application of a mitochondrial function intervention material in preparation of health-care products or medicines for improving mitochondrial functions. The result shows that the mitochondrial function intervention material can obviously improve the accumulation of acyl carnitine, obviously increase the basic oxygen consumption, ATP generation, maximum oxygen consumption and the like, and can be applied to the fields of health-care food, medicines and the like.
Compared with the existing evaluation screening system, the screening method of the mitochondrial function intervention material provided by the invention is more scientific, accurate and efficient. Compared with H2O2, the modeling of insulin and dexamethasone has the characteristics of high stability, obvious effect and high construction power, and can effectively simulate the mitochondrial damage caused by obesity and abnormal lipid metabolism. The method adopts the Seahorse real-time metabolism monitoring combined with the triple quadrupole composite linear ion trap liquid-mass combined technology, and accurately, quickly and intuitively detects and analyzes the respiratory metabolism condition of mitochondria and metabolites. The system combines WB, QPCR and mtDNA detection technologies, carries out further analysis on the intervention substances screened by the system, explores possible action mechanisms of the intervention substances from gene and protein levels, and further explores specific action mechanisms of functional components by detecting the ratio of ROS content, SOD content, MDA content and NAD +/NADH content and scanning electron microscope technology. The whole system forms a complete system from rapid screening to specific action mechanism exploration, and realizes scientific screening from appearance phenomenon, metabolite analysis to specific pathway exploration. Compared with the traditional in-vitro screening system and the animal screening experiment system, the system has the characteristics of high efficiency and science, can provide scientific and sufficient data support for later-stage animal and flora experiments, and greatly saves time and capital cost.
Drawings
FIG. 1 is a graph of the relative amounts of acylcarnitines in C2C12 cells after Palmitic Acid (PA) stimulation.
Fig. 2 is a graph showing the relative content of acylcarnitines after NMN treatment.
FIG. 3 is a graph showing the relative content of acylcarnitines after sea cucumber peptide (HST) treatment.
FIG. 4 is a graph showing the relative content of acylcarnitines after the aqueous extract of broccoli seeds was treated.
FIG. 5 is a graph showing the relative amounts of acylcarnitines after quercetin treatment.
FIG. 6 is a graph showing the relative content of acylcarnitines after treatment with an extract of Lycium barbarum.
FIG. 7 is a graph showing the relative content of acylcarnitines after hesperidin treatment.
FIG. 8 is a graph showing the result of the assay of Seahorse in the NMN and holothurian peptide (HST) group.
FIG. 9 is a graph showing the results of the assay of broccoli seed water extract, quercetin, and Lycium barbarum extract group Seahorse.
FIG. 10 is a graph showing the results of measurement of the quercitrin group Seahorse.
FIG. 11 is a graph showing the result of mtDNA measurement.
FIGS. 12 to 14 are graphs showing the results of real-time quantitative PCR.
FIG. 15 is a graph showing the results of western blot detection of proteins involved in the proliferation and oxidative phosphorylation of skeletal muscle cells.
FIG. 16 is a diagram showing the results of the measurement of the contents and ratios of ROS, SOD, MDA and NAD +/NADH.
Fig. 17 is a mitochondrial morphology analysis image.
Detailed Description
The present invention is further illustrated by the following specific embodiments, which are not intended to limit the scope of the invention.
First, experiment method
1. Cell culture
C2C12 cells (mouse myoblasts) are selected as the cell line;
cell growth medium: DMEM complete medium containing 10% FBS (fetal bovine serum), 1% P/S (double antibody, 100U/mL penicillin and 100. mu.g/mL streptomycin);
cell differentiation medium: DMEM complete medium contains 2% HS (horse serum), 1% P/S;
growth conditions are as follows: 37 ℃ and 5% CO2。
2. Cell passage
And (3) carrying out passage when the cell fusion rate reaches 80-85 percent:
(1) taking out the cells in the incubator, observing whether passage is needed or not under a microscope, putting the cells in an ultra-clean bench, then sucking out old culture solution in a culture dish, and adding 1-2 mL of PBS to wash the cells;
(2) adding 1mL of pancreatin to digest the cells, slightly shaking the culture dish to cover the pancreatin in the whole dish, observing by using a microscope, and removing the pancreatin when 50-70% of the cells become bright and round;
(3) adding 2mL of DMEM culture solution containing serum to stop pancreatin digestion, and blowing and beating the digested cells by a pipette to fall off from the bottom of the culture dish to form cell suspension;
(4) transferring the cell suspension to a 15 mL centrifuge tube, centrifuging at 700-;
(5) removing the supernatant, adding 2-3 mL of growth culture solution, and mixing by pipetting (small amplitude and high frequency) under the liquid level;
(6)1: transferring 5 passages into 10 cm cell culture dish (adding 8-9 mL of growth culture solution in advance) for
Culturing;
(7) and (4) covering the dish cover, pressing the edge of the dish, shaking uniformly in a cross shape, and having moderate degree (observing whether the cell distribution is uniform or not under a microscope, wherein the cell growth state is influenced by the nonuniformity).
3. Cell differentiation
C2C12 myoblasts are subjected to subsequent experiments and need to be differentiated into myotubes, and the cell morphology is to be the myotubes with fusiform structures;
(1) culturing cells with 6-well plate according to the above method, and sucking out the culture medium when the cell growth fusion rate is close to 80%
Differentiating by using a differentiation medium containing 2% HS instead of a growth medium containing 10% FBS;
(2) the differentiation medium is generally replaced once in 1 to 2 days, and obvious myotubes can be formed in the differentiation time of about 3 to 4 days according to the cell state.
4. Raw material treatment
(1) Mixing Nicotinamide Mononucleotide (NMN), Stichopus japonicus peptide (HST), water extract of broccoli seed, quercetin, and fructus Lycii
The extract and hesperidin are prepared at appropriate concentration, and the water soluble materials are dissolved overnight in serum-free culture medium. The fat-soluble raw materials are dissolved by adopting cell-grade DMSO, and the concentration of the DMSO in the final culture medium is not more than 0.5%;
(2) preparing a Palmitic Acid (PA) solution: PA 51.284 mg was weighed out and dissolved in 1mL of absolute ethanol to prepare a 200 mM solution. 800 mg of BSA (without fatty acids) were weighed out and dissolved in 39 mL of 0.01M PBS. 1mL of 200 mM PA was added to 39 mL of PBS to prepare a final concentration of 5mM PA solution, which was then filtered through a 0.22 μm filter and dispensed, and stored at-20 ℃ until use.
(3) Differentiation medium in C2C12 cells after 4 days of differentiation was blotted dry and rinsed with 2mL PBS, then PA solution and the previously prepared material were added. The final concentration of PA was made 1.6 mM. And (3) placing the cells added with the raw materials into a cell culture box for culturing for 24h, and then sucking cell culture fluid and storing at-80 ℃ for measuring the acyl carnitine.
5. Determination of acylcarnitines
The determination of acylcarnitines was performed by LC-MS/MS.
The cell culture broth was centrifuged (10000 rpm, 15 min, 4 ℃). Then 100 muL of supernatant is taken and dried in a 1.5 mL centrifuge tube under ultra-pure nitrogen. Then 100 μ L of chromatographic grade methanol was added, well mixed and then centrifuged (10000 rpm, 15 min, 4 ℃), and the supernatant liquid was aspirated for testing.
The standard samples were diluted to 500, 1000, 2000, 4000, 8000 respectively for preparing a standard curve.
The instrument is a triple four-rod composite linear ion trap liquid mass spectrometer of American AB Sciex company, model: QTRAP 5500. A chromatographic column: kinetex C18100A 100 x 2.1mm, 2.6 μm.
Information of the ion pair of the target analyte is determined and then the sample is assayed.
Mobile phase A: 5mM ammonium acetate water, B: acetonitrile, and the concentration calculated by substituting the data into a standard curve.
As a result, as shown in fig. 1, Palmitic Acid (PA) stimulation significantly increased the release of a fraction of acylcarnitines in C2C12 cells, particularly long-chain acylcarnitines such as octanoylcarnitine (C8), lauroylcarnitine (C12), myristoylcarnitine (C14), and palmitoylcarnitine (C16). Carnitine is an essential cofactor for the oxidative supply of long-chain fatty acids into the mitochondria, during which carnitine and the corresponding fatty acids are dehydroesterified to form acylcarnitines under the action of lipid acyltransferases. Carnitine and acyl-carnitine play important roles in cell energy distribution and the process of transporting fatty acids to metabolic oxidation sites in mitochondrial membranes. When mitochondria are damaged or overloaded, accumulation of long-chain acylcarnitines results. Therefore, the results show that the mitochondrial damage model constructed by PA is effective and feasible. As shown in FIGS. 2-7, NMN, Stichopus japonicus peptide, broccoli seed water extract, quercetin, fructus Lycii extract, and hesperidin can significantly reverse the accumulation of acylcarnitines caused by PA, especially the content of medium-long chain acylcarnitines.
6. Seahorse assay
Mito stress test kit was purchased from Seahorse Biosciences.
Preparation of detection Medium 100 mL of XF Base Medium was placed in a clean and oven-dried jar, and 0.1g of glucose (D-glucose) and 1.1 mL of 100 mM sodium pyruvate (sodium pyruvate) were added. Then, the pH value is adjusted to 7.4 by using 1N NaOH, and the solution passes through a 0.22 mu m filter membrane and is stored to be at the temperature of-4 ℃ for later use.
C2C12 cells were seeded at 5000/well concentration into Seahorse XF plates and growth medium was added the day before the experiment, and PA and starting material were added for 24h after the cells were attached at the concentration in the previous acylcarnitine experiment. Both PA and feed were prepared in serum-free DMEM medium.
Add Seahorse XF to Utility plate to calibrate 1 mL/well and make CO free2The probes were hydrated overnight in the incubator.
On the experimental day, the culture medium in the stimulated cell culture plate is sucked dry, 525 muL of detection culture medium is added, the detection culture medium needs to be preheated in advance in a 37 ℃ water bath kettle, and then the cell plate is put into a 37 ℃ CO-free container2Culturing in an incubator for 1 h.
The method comprises the steps of preparing medicines of oligomycin A (oligomycin), carbonyl cyanide-4-trifluoromethoxybenzenehydrazone (FCCP) and Rotenone/antimycin A (Rotenone/antimycin A), adding the medicines into a test board, and sequentially adding A, B, C medicine holes, wherein the adding amount of the three medicines in each hole is 75 mu L, and the final concentrations of the three medicines in a solution are 1 mu M, 0.5 mu M and 0.5 mu M respectively.
A total of 24 wells in the cell plate, of which four wells a 1B 4C 3D 6 were control wells, and no cells were added. The PA group was set as a control for each experiment and used for final data normalization.
The primed test plate and overnight hydrated Utility plate instrument were placed on the instrument tray and the procedure was run and the cell plate was replaced after about 30 minutes.
The results are shown in fig. 8-10, and compared with the PA group, the NMN, the sea cucumber peptide, the broccoli seed water extract, the quercetin, the wolfberry extract, and the hesperidin group have a significant improvement effect on the mitochondrial function OCR curve, and significantly increase the basal oxygen consumption, ATP generation, and maximum oxygen consumption.
7. mtDNA assay
After the skeletal muscle cells of the C2C12 mouse are cultured and differentiated for 4 days, stimulation treatment is carried out according to the screened test substance, and the treatment is carried out for 24 hours
Then, DNA in muscle cells and mitochondria is respectively extracted by using the kit, and primers required to be measured are synthesized. Quantitative PCR detection of mitochondrial DNA content: a PCR reaction solution was prepared with the following composition.
DNA template 0.5 μ L;
2X Master Mix 10 μL；
8.5 mu L of sterilized distilled water;
PCR forward and reverse primers total 1. mu.L;
Total 20 μL/Sample；
the PCR reaction assay was performed and the samples were stored at 4 ℃ after the reaction. Each sample was provided with 3 duplicate wells, and all experiments were repeated
6 times. Copy number was obtained by quantitative PCR, and the relative value of mitochondrial DNA content (mitochondrial DNA/genomic DNA) in the cells was calculated. The results are shown in fig. 11, PA significantly reduced mitochondrial mtDNA, which further demonstrates that PA disrupts mitochondrial DNA, resulting in reduced mitochondrial function, which NMN and sea cucumber peptides can significantly reverse.
8. Real-time quantitative PCR
After the skeletal muscle cells of the C2C12 mouse are cultured and differentiated for 4 days, stimulation treatment is carried out according to the screened test substance, and the treatment is carried out for 24 hours
Then, Trizol collects cells, extracts RNA, and reverse transcribes to prepare cDNA. The PCR reaction system was prepared as follows:
a conventional qPCR composition 10. mu.L system;
2 x PowerUp SYBR Green Master Mix（ABI） 5μL；
the total amount of the forward primer and the reverse primer is 0.6 mu L;
cDNA template and ddH2O 4.4.4. mu.L;
the total volume is 10 mu L;
the mRNA level expression of 27 genes mainly related to mitochondria is detected, and the specific names of the genes are as follows:
ClpP、HSP60、PPARD、CYTC-1、COX1、mtATP8、mtCOX2、SDHA、SOD1、SOD2、PGC1a、SIRT1、NRF1、NRF2、FIS1、Acox1、Acadl、PGC1b、CPT1b、OPA1、TFAM、MFN1、MFN2、NDUFS1、UCP2、ATP5g1、COX4、36b4、B2m、ERRa、Drp1。
as shown in fig. 12-14, PA treatment affected the expression of mitochondria-associated genes in C2C12 cells to some extent, but fold-changes were all within 5-fold, compared to serum-free DMEM blank; after NMN and sea cucumber peptide treatment, most of the mitochondria-related genes changed significantly, especially PPARD, Sirt3, OPA1, HSP60 and CPT1b, and mRNA expression appeared to be increased hundreds of times.
9. Detection of mitochondria-related gene and oxidative phosphorylation complex protein western blot in skeletal muscle
After the skeletal muscle cells of the C2C12 mouse are cultured and differentiated for 4 days, stimulation treatment is carried out according to the screened test substance, and the treatment is carried out for 24 hours
After that, cells were collected with RIPA lysate to prepare protein samples, and then western blot analysis was performed by SDS-PAGE to detect protein level expression of mitochondrial key genes such as oxidative phosphorylation complex (OXPHOS), Mitochondrial Transcription Factor (MTFA), cytochrome C, mitochondrial fusion protein 1(OPA1), and motility-related protein 1 (DRP 1).
As shown in FIG. 15, PA stimulation significantly reduced the protein expression level of Cytc-1 in cells, while NMN and Stichopus japonicus peptide treatment increased the protein level of Cytc-1 in C2C12 cells, and there was a tendency for respiratory chain complex I to increase.
10. Detecting the content of ROS, SOD, MDA and NAD +/NADH and the proportion
After the skeletal muscle cells of the C2C12 mouse are cultured and differentiated for 4 days, stimulation treatment is carried out according to the screened test substance, and the treatment is carried out for 24 hours
And then, discarding the culture solution, adding cold Phosphate Buffer Solution (PBS) into the cells, collecting the cells by using a cell scraper, then performing lysis analysis by using a related kit, finally reading corresponding light absorption values, and calculating the relative contents of ROS, SOD, MDA and NAD +/NAD by taking the total cell protein concentration as a standard.
As shown in fig. 16, both NMN and sea cucumber peptide can reduce ROS excess caused by PA and MDA overexpression, and activate SOD expression, and at the same time, NMN can significantly increase the ratio of NAD +/NAD.
11. Mitochondrial morphology analysis (transmission electron microscopy)
After the skeletal muscle cells of the C2C12 mouse are cultured and differentiated for 4 days, stimulation treatment is carried out according to the screened test substance, and the treatment is carried out 24
After an hour, the culture solution was aspirated, a cold Phosphate Buffer Solution (PBS) was added to the cells, the cells were harvested by a cell scraper, and then centrifuged at 700rpm for 5 minutes at 4 ℃ to obtain cell pellets, freshly prepared precooled 25Ml/L glutaraldehyde-fixed cells were added, 1% (mass fraction) of OsO4 was fixed again, and after ethanol gradient dehydration, acetone, propylene oxide treatment, embedding with epoxy resin 812, positioning under a microscope to take a site repair mass with a large number of cell colonies, parallel longitudinal ultrathin sections (70nm) with reference to a PET polyester film calibration plane, staining with uranyl acetate and lead citrate, and the ultrastructure of cell mitochondria was observed and photographed under a JEM-1400 transmission electron microscope (Japan Electron Co., Ltd., Japan) under 80 kV.
Mitochondrial morphology analysis images show that as shown in fig. 17, PA treatment causes swelling and volume enlargement of part of mitochondria, and internal ridge structure is destroyed, resulting in disorder of mitochondrial structure; NMN and sea cucumber peptide treatment can reverse damage of PA to mitochondria, so that the mitochondria volume is reduced, the mitochondria number is increased, the ridge structure is recovered to be normal and clearly visible, and the mitochondria morphology tends to be normal.

Claims (8)

1. The application of the mitochondrial function intervention material in preparing health care products or medicines for improving mitochondrial functions is characterized in that the mitochondrial function intervention material is one or a mixture of more of NMN, holothurian peptide, broccoli seed water extract, medlar extract, quercetin, hesperidin or centella asiatica extract, and preferably NMN or holothurian peptide.
2. The use of claim 1, wherein the improvement in mitochondrial function is improvement in accumulation of acyl carnitine.
3. The use of claim 1, wherein the improvement in mitochondrial function is improvement in basal oxygen consumption, ATP production, or maximal oxygen consumption.
4. A method for screening a mitochondrial function intervention material is characterized by comprising the following steps:
(1) constructing a mitochondrial damage model by using palmitic acid;
(2) determining acylcarnitine by an LC-MS/MS method;
(3) detecting the respiratory function by using a Seahorse extracellular flux analyzer;
(4) detecting the content of mitochondrial DNA by quantitative PCR;
(5) real-time quantitative PCR detection of mRNA level expression of genes mainly related to mitochondria;
(6) detecting related genes and oxidative phosphorylation complex proteins of mitochondria in skeletal muscle by using a Western blot method;
(7) detecting the MDA content and the NAD +/NADH content by using a kit;
(8) and observing the ultrastructure of cell mitochondria by adopting a transmission electron microscope and carrying out morphological analysis.
5. The method for screening mitochondrial function intervention materials according to claim 4, wherein the step (1) of constructing the mitochondrial damage model by using palmitic acid specifically comprises the following steps:
a. cell culture
Selecting C2C12 myoblasts, and putting the myoblasts into a growth culture medium for culture;
b. cell passage
Sucking out the growth medium when the cell fusion rate reaches 80-85%, washing the cells with PBS, adding pancreatin to digest the cells, and adding trypsin when the cell fusion rate reaches 80-85%
When 50-70% of cells become bright and round, absorbing pancreatin, adding a growth culture medium to stop pancreatin digestion, shedding digested cells to form a cell suspension, centrifuging, absorbing a supernatant, adding the growth culture medium, and transferring the cell suspension to a culture dish added with the growth culture medium according to a specific passage ratio for culture;
c. cell differentiation
Sucking out the growth medium when the cell growth fusion rate reaches 75-80%, and adding a differentiation medium for differentiation;
d. raw material treatment
Sucking up a differentiation culture medium in differentiated C2C12 myoblasts, rinsing with PBS, adding a palmitic acid solution and a raw material, culturing the cells added with the raw material in a cell culture box, sucking a cell culture solution, and storing at-80 ℃ for later use.
6. The method for screening mitochondrial function intervention material of claim 5, wherein the growth medium comprises 10% FBS and 1% P/S; the differentiation medium contained 2% HS and 1% P/S.
7. The method for screening mitochondrial function intervention material according to claim 5, wherein the cell culture conditions are 37 ℃ and 5% CO2。
8. The method for screening mitochondrial function intervention materials according to claim 5, wherein the palmitic acid solution is prepared by:
weighing palmitic acid, and dissolving the palmitic acid in absolute ethyl alcohol to obtain a palmitic acid ethanol solution; weighing BSA and dissolving in PBS to obtain a PBS solution; the ethanol palmitic acid solution was added to the PBS solution to prepare a palmitic acid solution having a final concentration of 5 mM.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115317475A (en) * 2022-08-22 2022-11-11 华中科技大学同济医学院附属协和医院 Application of quercetin in preparation of medicine for preventing or treating metabolism-related fatty liver disease

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016726A2 (en) * 2002-08-09 2004-02-26 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
US20140065099A1 (en) * 2011-02-15 2014-03-06 Ecole Polytechnique Federale De Lausanne (Epfl) Methods of Treating Mitochondrial Dysfunction
CN103720685A (en) * 2014-01-14 2014-04-16 南京大学 Application of quercetin in preparation of medicines for treating mitochondrial dysfunction related diseases
WO2020012175A1 (en) * 2018-07-10 2020-01-16 Nuchido Limited Compositions that protect cells from oxidative and mitochondrial stress
CN111374310A (en) * 2020-04-16 2020-07-07 上海益跬尔生物科技有限公司 Edible compound mitochondrial energy enhancing formula

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004016726A2 (en) * 2002-08-09 2004-02-26 President And Fellows Of Harvard College Methods and compositions for extending the life span and increasing the stress resistance of cells and organisms
US20140065099A1 (en) * 2011-02-15 2014-03-06 Ecole Polytechnique Federale De Lausanne (Epfl) Methods of Treating Mitochondrial Dysfunction
CN103720685A (en) * 2014-01-14 2014-04-16 南京大学 Application of quercetin in preparation of medicines for treating mitochondrial dysfunction related diseases
WO2020012175A1 (en) * 2018-07-10 2020-01-16 Nuchido Limited Compositions that protect cells from oxidative and mitochondrial stress
CN111374310A (en) * 2020-04-16 2020-07-07 上海益跬尔生物科技有限公司 Edible compound mitochondrial energy enhancing formula

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
B.VIJAYABHARATHI等: "Protective Effects of Hesperidin (CitrusFlavonone) On High Glucose Induced Oxidative Stress and Apoptosis in a Cellular Model for Diabetic Retinopathy", 《ANDROLOGIA》 *
LARYSAV.YUZEFOVYCH等: "Protection from Palmitate-Induced Mitochondrial DNA Damage Prevents from Mitochondrial Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and Impaired Insulin Signaling in Rat L6 Skeletal Muscle Cells", 《ENDOCRINOLOGY》 *
于雁飞: "海参肽对运动性疲劳大鼠骨骼肌线粒体功能的影响", 《山东医药》 *
何珺等: "12种十字花科蔬菜种子中萝卜硫素含量研究", 《食品研究与开发》 *
姚晓黎等: "枸杞提取物对 MPP + 诱导的线虫和 PC12细胞神经毒性的保护作用", 《中药材》 *
熊御云等: "积雪草酸抗帕金森样神经损伤的线粒体机制", 《2013全国老年性痴呆与相关疾病学术会议》 *
王欢: "NMN通过 NAD+/Sirt3 通路改善线粒体功能抑制间充质干细胞衰老的作用研究", 《中国优秀硕士学位论文全文数据库 (基础科学辑)》 *
蒲道静: "萝卜硫素干预纳米细菌诱导肾小管上皮细胞凋亡及线粒体自噬相关蛋白的表达", 《中国组织工程研究》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115317475A (en) * 2022-08-22 2022-11-11 华中科技大学同济医学院附属协和医院 Application of quercetin in preparation of medicine for preventing or treating metabolism-related fatty liver disease

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