CN110656083A - Pre-adipocyte brown induction kit - Google Patents
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Abstract
The invention discloses a preadipocyte brown induction kit, which comprises: buckwheat hull flavone, insulin, dexamethasone, 3-isobutyl-1-methylxanthine; a method for inducing browning of preadipocytes, comprising: 1) inoculating preadipocytes, culturing until the preadipocytes are completely converged, and continuously inhibiting for 2 days; 2) sucking out cells, adding buckwheat husk flavone into differentiation culture medium
Culturing for 2 days, adding buckwheat husk flavone into differentiation culture medium
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a preadipocyte browning induction kit.
Background
Obesity and diabetes have become global health problems that seriously harm people's health in modern society and have become the causes of various diseases. Obesity is a main risk factor for the occurrence and development of IR and T2DM, and obesity, IR and T2DM have complex causal relationship, correlation and interdependence. The obese people usually have complicated metabolic disorders, which can easily cause metabolic syndromes such as insulin resistance, T2DM, hyperinsulinemia, hyperlipidemia, hypertension, cardiovascular and cerebrovascular diseases, non-alcoholic fatty liver disease, cancer and the like, and serious diseases. Statistically, the incidence of obesity and diabetes and its complications is rapidly increasing, with 80% of type 2 diabetic patients being accompanied by obesity, which is predicted to reach 6 billion worldwide by 2035. Most of the medicines for treating T2DM and insulin in the current market have serious toxic and side effects, and can cause obesity of the body while improving blood sugar. However, the research shows that the bioactive substances of the commonly used medicinal and edible plants have good improving effect on T2DM and metabolic disorders related to insulin resistance, and the toxic and side effects on the body are remarkable, and the functions of the bioactive substances are diversified.
Fat plays a key role in systemic metabolism and energy homeostasis. Two main types of adipose tissue are present in the human body: white adipose tissue and brown adipose tissue. White Adipose Tissue (WAT) is mainly dispersed in the tissues of omentum, perirenal membrane, mesentery, peritoneum and subcutaneous tissue in the abdominal cavity, and is used exclusively for storing energy in the form of triglycerides, and is also involved in endocrine signaling and metabolic immune crosstalk, the main function of which is to supply energy for human life activities. Meanwhile, when the energy of the human body is excessive for a long time, lipid is excessively accumulated, and obesity is caused. In obese individuals, white adipocytes have reduced oxygen consumption, thermogenic activity, and mitochondrial content. In contrast, brown adipocytes, which are filled with mitochondria, play an important role in preventing hypothermia, increasing energy expenditure, and improving insulin sensitivity. Brown Adipose Tissue (BAT) begins to form during fetal life and peaks in infancy, but gradually degrades and decreases in number with age, body development, and Brown adipose tissue function. Adult BAT is mainly distributed in the regions of active metabolism, such as the clavicle of the neck, the interscapular region, the spine, the groin, the peripheral vascular region, the perirenal region, and the epicardium. BAT is brown due to its abundance in hemoglobin and its considerable content of cytochromes. Unlike WAT, its primary function is heat production. Many studies have shown that BAT plays an important role in systemic energy homeostasis, substrate metabolism and glucose homeostasis. For example, BAT transplantation can significantly reduce body weight, improve glucose metabolism and insulin sensitivity in mice fed a high fat diet. Suggesting that BAT may be targeted for the treatment of obesity related diseases such as insulin resistance, metabolic syndrome and diabetes [12 ]. Recent studies have found that another fat exists in the human body, which is derived from white fat, but has the appearance and function of brown fat, called Beige edipose tissue (BeAT). The thermogenic activity of brown and beige adipocytes can be used to reduce energy surplus and counteract obesity. Recent studies have shown that beige adipocytes are indeed present in adults, and recruitment of beige adipocytes can accelerate body energy expenditure and maintain glucose homeostasis, so that it is increasingly appreciated that beige adipocytes may be a therapeutic target for obesity and obesity-related diseases, including type 2 diabetes. To date, transcriptional control of brown and beige adipocyte formation has been extensively studied.
Buckwheat belongs to the genus Fagopyrium (Polygonaceae), but is a small grain, and thus has a low yield and low consumption compared to a large grain, and has not been sufficiently developed. Buckwheat and its by-products also contain many functional active ingredients, such as flavonoids, buckwheat alkali, chiro-inositol, buckwheat sugar alcohol, functional proteins, etc. Clinical tests show that the buckwheat can improve the incidence of diabetes after being eaten for a long time, and has better prevention and improvement effects on cardiovascular and cerebrovascular diseases, hypertension, hyperlipidemia, obesity and cancers caused by lipid metabolism disorder. The buckwheat hulls are important byproducts in buckwheat processing, part of the buckwheat hulls are used as fillers of fragile goods and pillow cores, or used as fuel, the added value is low, most of the buckwheat hulls are directly discarded, effective and full utilization is not achieved, and great resource waste is caused. Researches show that the waste buckwheat hulls are rich in flavonoids, such as rutin, isoquercitrin, isoorientin, orientin, vitexin, quercetin, (quercetin 3-D-glucoside) and isovitexin, and the like, and the buckwheat hulls contain different functional components due to different buckwheat varieties and different planting areas. The research finds that the sweet buckwheat hull flavone can remarkably promote glucose absorption and lipid synthesis and accelerate lipid decomposition, and the mechanism of the sweet buckwheat hull flavone is closely related to fat browning.
Disclosure of Invention
The invention aims to provide a preadipocyte browning induction kit.
A preadipocyte browning induction kit, comprising: buckwheat hull flavonoids;
the preparation method of the buckwheat hull flavone comprises the following steps:
1) drying testa Fagopyri Esculenti, pulverizing, adding water, and extracting at high temperature under high pressure;
2) filtering, purifying the filtrate with macroporous resin, concentrating, and lyophilizing to obtain buckwheat hull flavone;
the macroporous resin is D101 and AB-8 type macroporous resin.
A method for inducing browning of preadipocytes, comprising:
1) inoculating preadipocytes at 37 deg.C with 5% CO2Culturing under the condition until preadipocytes are completely confluent, and continuously inhibiting for 2 days;
2) sucking out the cultured cells in the step 1), and adding buckwheat hull flavone into the differentiation culture medium
Culturing for 2 days, adding buckwheat husk flavone into differentiation culture medium
Culturing for 2 days in medium culture medium, and culturing the buckwheat husk flavone in differentiation culture mediumAnd
the final concentration of (5) is 10 ~ 100 mug/mL;
3) then, continuously culturing by using a complete culture medium containing 10% FBS, replacing the culture medium every 2 days until 90% of cells show adipocyte phenotype, namely when lipid drops accumulate, the cells are completely differentiated, and finishing the culture;
the components of the culture medium are respectively as follows:
1) complete culture medium 40 ~ 50mL DMEM, 3 ~ 10mL FBS, 450 ~ 550mL penicillin-streptomycin mixed solution;
2) differentiation mediumDMEM 48.8 ~ 49.2.2 mL, 0.5 mM 3-isobutyl-1-methyl xanthine 450 ~ 550 muL, 10 mug/mL insulin 45 ~ 55 muL and 1 muM dexamethasone 45 ~ 55 muL containing 10% FBS and 1% double-antibody respectively in volume fraction, and adding DMEM to supplement the volume fraction to 50 mL;
3) differentiation medium
DMEM 48.8 ~ 49.2.2 mL containing 10% FBS and 1% double-antibody respectively in volume fraction, and 150 ~ 250 mu L of 10 mu g/mL insulin is added to make up to 50 mL;
the preadipocytes are 3T3-L1 preadipocytes;
the buckwheat hull flavone is in a differentiation culture medium
And
the final concentration of (5) is 10 ~ 80 mug/mL;
the buckwheat hull flavone is in a differentiation culture medium
Andthe final concentration in (a) is 50 mug/mL.
The invention provides a preadipocyte brown induction kit, which comprises: buckwheat hull flavonoids; a method for inducing browning of preadipocytes, comprising: 1) inoculating preadipocytes at 37 deg.C with 5% CO2Culturing under the condition until preadipocytes are completely confluent, and continuously inhibiting for 2 days; 2) sucking out the cultured cells in the step 1), and adding buckwheat hull flavone into the differentiation culture medium
Culturing for 2 days, adding buckwheat husk flavone into differentiation culture medium
Culturing for 2 days in medium culture medium, and culturing the buckwheat husk flavone in differentiation culture medium
And
the final concentration of the buckwheat husk flavone PBHE-M4 is 10 ~ mug/mL, 3) the culture is continued by using a complete culture medium containing 10% FBS, the culture medium is replaced once every 2 days until 90% of cells show adipocyte phenotype, namely when fat drops accumulate, the cells are completely differentiated, and the culture is finished.
Drawings
FIG. 1 results of cytotoxicity of buckwheat hull flavonoids on 3T3-L1 preadipocytes;
FIG. 23 is a schematic representation of the "cocktail" induced differentiation of preadipocytes from T3-L1;
FIG. 3 Effect of buckwheat hull flavonoids on cellular activity during differentiation of 3T3-L1 preadipocytes;
FIG. 4 Effect of buckwheat husk flavone on the differentiation of 3T3-L1 pre-adipocytes (400 x);
FIG. 5 Effect of buckwheat hull flavonoids on 3T3-L1 pre-adipocyte lipid accumulation;
FIG. 6 Effect of buckwheat chaff flavone on differentiation of 3T3-L1 pre-adipocytes in the presence of rosiglitazone (400 x);
FIG. 7 effect of buckwheat hull flavone and rosiglitazone on lipid accumulation during differentiation of 3T3-L1 preadipocytes;
FIG. 8 Effect of buckwheat hull flavonoids on glucose uptake (a), triglyceride content (b), and glycerol release (c) by 3T3-L1 preadipocytes;
FIG. 9 KEGG Pathway (a) enrichment results; (b) analyzing the number of differentially expressed genes;
FIG. 10 is a clustering analysis of the Thermogenesis Pathway and oxidative phosphorylation expression difference-associated gene analysis (a, c); (b, d) differentially expressing the genes.
Detailed Description
Example 1 buckwheat hull flavones (PBHE, PBHE-M)4) Preparation of
The buckwheat hulls are purchased from the buckwheat rice factory Hongyan of Nenggu Chifeng city; extracting Buckwheat hull flavone extract (BHE) with hot water under high pressure to obtain total flavone, specifically: drying and crushing buckwheat hulls, weighing 500g of buckwheat hull powder, adding water, and mixing the mixture in a ratio of 1: 15, carrying out hot water high-pressure leaching for 2 times, each time for 60min, concentrating and freeze-drying the leaching liquor, purifying by using D101 macroporous resin, centrifuging and evaporating at 75 ℃ under reduced pressure, and then carrying out vacuum freeze-drying to obtain the BHE. After washing the unabsorbed compound with distilled water, elution was performed with 70% ethanol at a flow rate of 6mL/min until no color was observed. The freeze-dried eluate was used for Purification of Buckwheat Husk Extract (PBHE) samples; then, re-purifying the PBHE by using AB-8 macroporous resin (Donghong chemical company, Shanghai, China); eluting the column with distilled water and ethanol of different concentrations according to the color change of the eluent; elution with 40% ethanol followed by lyophilization yielded a sample of PBHE-M4. The content of flavone is determined by taking rutin as a standard substance, and the content of total flavone of PBHE and PBHE-M4 is 74.79% and 89.92% respectively. The flavonoid compounds in PBHE and PBHE-M4 include rutin, vitexin, isoorientin and hyperoside.
Example 2 toxicity test of buckwheat husk flavone on 3T3-L1 preadipocytes
Determining the cytotoxicity of a sample to be detected on cells by adopting an MTS method, aiming at determining the optimal intervention concentration; 3T3-L1 preadipocytes at 5X 103Inoculating each well (100 μ L) to 96-well plate at 37 deg.C and 5% CO2And incubating for 24h under the saturated humidity condition; after the cells are confluent, the culture medium is removed and replaced with buckwheat husk flavone (PBHE, PBHE-M) of different concentrations4) For 48h, the concentration was set as: 10. 20, 40, 80, 100 mug/mL; removing the culture medium, adding 100 muL culture medium into each well, adding 20 muL MTS, and culturing at 37 deg.C with 5% CO2Incubating for 1-4h in the environment, reading the absorbance value at 512nm by using an enzyme-labeling instrument, and simultaneously measuring the absorbance value at 700 nm to remove the background value caused by excessive cell debris;
the complete culture medium formula is as follows: 45 mL of DMEM + 5 mL of FBS + 500mL of penicillin-streptomycin mixed solution;
the results are shown in FIG. 1, PBHE and PBHE-M4Has no obvious influence on cell proliferation under the concentration of 10-80 mug/mL and is 100 mug/mL PBHE-M4The influence on the cells is large, so that safer 50 mug/mL concentration is selected for carrying out subsequent intervention experiments.
Example 3 Induction of differentiation by T3-L1 Pre-adipocytes
3T3-L1 preadipocytes were induced to differentiate using the classical "cocktail" method, as shown in FIG. 2. According to the experimental requirements, 3T3-L1 preadipocytes are inoculated into corresponding culture bottles or culture plates according to certain density and are cultured at 37 ℃ in 5% CO2Culturing in a saturated humidity incubator, culturing for 2-3 days by using a complete culture medium, replacing a differentiation culture medium containing 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 mu M Dexamethasone (Dexamethasone, DXMS) and 10 mu g/mL Insulin (Insulin, INS) after the preadipocytes are completely confluent and continuously contact and inhibit for 2 days
Culturing for 2 days (0-2 days), and changing with differentiation culture medium containing 10 mug/mL INS
Cultured for 2 days (2-4 days). On day 4, culture was continued in complete medium containing 10% FBS, and the medium was changed every 2 days until day 8, when 90% of the cells exhibited adipocyte phenotype, i.e., lipid droplets were accumulated, the cells were completely differentiated and used for the relevant index test. To study the effect of buckwheat hull flavone component on differentiation of 3T3-L1 preadipocytes, buckwheat hull flavone (PBHE, PBHE-M) was added on days 0-4, respectively4) Added to differentiation media I and II at a concentration of 50 μ g/mL (diluted with media to a final concentration of 50 μ g/mL, example: 24-well plates with 1.25. mu.L to 500. mu.L of medium per well);
the components of the culture medium are as follows:
1) complete medium: 45 mL of DMEM, 5 mL of FBS and 500mL of penicillin-streptomycin mixed solution;
2) differentiation medium
: 49 mL of DMEM containing 10% FBS and 1% double-antibody, 500 muL of 0.5 mM 3-isobutyl-1-methyl xanthine, 50 muL of 10 mug/mL insulin and 50 muL of 1 muM dexamethasone are contained in volume fraction, and 400 muG LDMEM is added to supplement the volume fraction to 50 mL;
3) differentiation medium
: 49 mL of DMEM containing 10% FBS and 1% double-antibody and 200 mu L of 10 mu g/mL of insulin are respectively contained, and 800 mu L of DMEM is added to supplement to 50 mL.
Example 4 Effect of buckwheat husk flavone on differentiation of 3T3-L1 Pre-adipocytes
3T3-L1 preadipocyte induced differentiation method reference example 3;
1. influence of buckwheat husk flavone on cell activity change during differentiation of 3T3-L1 preadipocytes
To study the effect of buckwheat hull flavone component on differentiation of 3T3-L1 preadipocytes, buckwheat hull flavone (PBHE, PBHE-M) was added on days 0-4, respectively4) Adding the cells into differentiation culture media I and II to enable the final concentration to be 50 mug/mL; 3T3-L1 preadipocytes required a 2 day contact inhibition period before induction of differentiation, during which time the cells were fully inhibited and cell activity was subsequently reduced. After 2 days of inhibition, buckwheat husk flavone (PBHE, PBHE-M) was added4) The results of the intervention are shown in FIG. 3. As can be seen from the figure, PBHE group and PBHE-M4The cell activity of the group was not significantly different from that of the Control group (p>0.05), thus indicating that buckwheat hull flavone does not affect cell activity during the differentiation induction of 3T3-L1 preadipocytes.
2. Effect of buckwheat husk flavone on differentiation of 3T3-L1 preadipocytes
1) Oil red O staining experiment
3T3-L1 preadipocyte induced differentiation method reference example 3;
3T3-L1 preadipocytes were inoculated into 24-well plates and after differentiation was complete, staining was performed with oil red O staining solution. Discarding the cell culture medium, washing twice with PBS buffer solution, adding 4% polyformaldehyde, fixing at room temperature for 30min, discarding the fixing solution, washing three times with PBS, and staining with oil red O solution for 10 min. Excess oil red O dye was washed 2-5 times with distilled water until there was no excess staining solution, and distilled water was added to cover the cells and pictures were taken under microscope.
The results of previous studies show that 3T3-L1 preadipocytes are influenced by various influencing factors, the fat differentiation rate is not high, and 90% fat phenotype is not achieved, as shown in FIG. 4(a, b); in previous in vivo experiments, buckwheat husk flavone was combined with STZ-induced T2DM rats and rats on high-fat dietdb/dbThe mice have good lipid-lowering effect, and can obviously reduce the expression of liver adipogenic genes, so that the buckwheat hull flavone can inhibit the differentiation of fat cells; however, it is apparent from the figure that buckwheat hull flavonoids are able to promote fat differentiation, which is inconsistent with previous experimental results.
2) Measurement of triglyceride content
The 3T3-L1 preadipocytes were seeded into 12-well plates, after differentiation was complete, the cells were washed twice with ice-cold PBS, lysed with lysate and TG concentration was determined directly according to Triglyceride (TG) kit determination. The total cellular protein concentration was measured using the BCA protein assay kit for intracellular TG content normalization.
On day 8 of cell differentiation, cells were lysed and collected, and Triglyceride (TG) accumulation after cell differentiation was examined using a triglyceride assay kit, as shown in FIG. 5. Buckwheat hull flavone can promote differentiation of 3T3-L1 preadipocytes, PBHE group and PBHE-M4The accumulation of TG cohorts was significantly higher than Model cohorts (without intervention). Besides, in the high flavone component PBHE-M4In the presence of 3T3-L1 preadipocytes, which lack IBMX, DXMS and INS in differentiation medium I and differentiation medium II, respectively, 3T3-L1 preadipocytes also appeared differentiated to different extents, wherein the accumulation of intracellular TG was significantly higher in the absence of INS than in the Normal group (no inducer treatment) ((R))p <0.01). Thus, buckwheat hull flavonoids have been shown to promote differentiation of 3T3-L1 preadipocytes.
Example 5 synergistic Effect of buckwheat husk flavone and rosiglitazone on 3T3-L1 Pre-adipocyte differentiation
3T3-L1 preadipocyte induced differentiation the specific method was as described in example 3 and slightly modified. The test groups were divided into a Normal group, a Rosi group (2 mM Rosi), a PBHE group (2 mM Rosi + 50 μ g/mLPBHE) and PBHE-M4Group (2 mM Rosi + 50 μ g/mL PBHE-M4). Respectively reserving cell culture fluid for inducing differentiation for 2 nd, 4 th, 6 th and 7 th days, centrifuging at 1500 rpm for 10min, collecting supernatant, and respectively filling the supernatant into 200 mu L centrifuge tubes, freezing and storing at-20 ℃, and performing next glucose uptake measurement after differentiation is finished. And (4) detecting the glucose content after diluting twice.
As a result: it is known from fig. 4 that buckwheat hull flavone has a significant effect of promoting differentiation of 3T3-L1 preadipocytes, but the differentiation rate thereof is less than 50%. Rosiglitazone, a thiazolidine antidiabetic drug, is a highly selective and potent agonist of PPAR γ, and can promote the expression of PPAR γ gene, thereby promoting the differentiation of 3T3-L1 pre-fat. As shown in FIG. 6 (a, b), in PBHE and PBHE-M4After 2 mu M rosiglitazone is added into the group, the differentiation rate and differentiation rate of the fat cells are greatly improved, and on the 6 th day of induction, PBHE + Rosi and PBHE-M4The + Rosi group achieved an effect of 8 days of induced differentiation before (without addition of rosiglitazone), and the drop of the atrial lipids was more pronounced. Culture was continued and on induction day 6, the lipid droplets began to gradually disappear. Fig. 7 (a, b) again demonstrates the synergistic effect of buckwheat hull flavone and rosiglitazone, and the 3T3-L1 preadipocyte differentiation rate with the co-intervention of buckwheat hull flavone and rosiglitazone was significantly higher than that with the dry pre-treatment group with buckwheat hull flavone and rosiglitazone alone. From fig. 8, it can be seen that the same results were observed, and the content of TG in adipocytes that intervened with the addition of buckwheat hull flavone and rosiglitazone was significantly higher at day 6 of differentiation than in Rosi dried group alone, and there was no significant change in TG content in Rosi group until day 8 from day 6: (p>0.05), but significant reduction in TG in cells with co-intervention of buckwheat husk flavone and rosiglitazone (p<0.05). Thus, the buckwheat hulls can synergistically promote the differentiation of fat cells, accelerate the differentiation process of 3T3-L1 preadipocytes and treat the cellsAfter complete differentiation, buckwheat hull flavone can promote lipid decomposition of TG without causing excessive accumulation of lipid.
Example 6 Effect of buckwheat husk flavones on glucose uptake, triglyceride content and glycerol release in 3T3-L1 adipocytes
1. Method of producing a composite material
Since PBHE-M4 was found to interfere better than PBHE in previous experiments, only PBHE-M4 was used for subsequent induction experiments. 3T3-L1 preadipocyte induced differentiation the specific method was as described in example 3 and slightly modified. The test groups were divided into a Normal group, a Rosi group (2 mM Rosi), and a PBHE-M4 group (2 mM Rosi + 50 μ g/mL PBHE-M4). Respectively reserving cell culture fluid for inducing differentiation for 2 nd, 4 th, 6 th and 8 th days, centrifuging at 1500 rpm for 10min, collecting supernatant, and respectively filling the supernatant into 200 mu L centrifuge tubes, freezing and storing at-20 ℃, and performing next glucose uptake and glycerol release measurement after differentiation is finished; before glycerol detection, extinguishing lipase at 70 deg.C for 10min, centrifuging at room temperature 5000 rpm for 5 min, and collecting supernatant for enzymology determination; and (4) detecting the glucose content after diluting twice.
2. Effect of buckwheat husk flavone on glucose uptake by 3T3-L1 preadipocytes
Insulin resistance is directly expressed as impaired biological response performance of target organs or target cells (such as liver, skeletal muscle, adipose tissue and the like) of an organism to endogenous or exogenous insulin, so that insulin sensitivity is reduced, and normal biological response concentration is not enough to play a role in regulating blood sugar, so that the absorption capacity of glucose is weakened; therefore, the improvement effect of buckwheat hull flavone on insulin resistance can be reflected by studying the glucose intake of fat cells. In this test, glucose uptake by 3T3-L1 preadipocytes was evaluated by measuring the glucose concentration in the culture broth, and the results are shown in FIG. 8 (a); during the differentiation of 3T3-L1 preadipocytes, the glucose uptake capacity of adipocytes was different at different time periods, and the glucose demand of each experimental group was the greatest at days 2-4 of induced differentiation, in which the glucose content in the culture broth of Rosi group was significantly reduced (p < 0.01) compared to that of Normal group. At the subsequent 4-6 days, the glucose demand of the cells in the Rosi group is gradually reduced, and the cells have no significant difference from the Normal group (p > 0.05); but the glucose content in the culture solution of PBHE-M4+ Rosi group was significantly reduced compared to the Rosi group (p < 0.0001). Therefore, the buckwheat hull flavone can promote the glucose uptake and utilization of 3T3-L1 preadipocytes, and has certain improvement effect on impaired insulin sensitivity and insulin resistance.
3. Effect of buckwheat husk flavone on triglyceride content of 3T3-L1 preadipocytes
The main functions of Triglyceride (TG) are energy storage and energy supply, and when TG exerts its energy supply function, TG needs to be hydrolyzed by triglyceride lipase to generate glycerol and free fatty acid, which are released into blood for organism tissue to utilize. The content of glycerol in the cell culture broth can reflect the level of TG lipolysis to some extent. As shown in the graph (b), after the buckwheat hull flavone intervenes for 2,4, 6 and 8 days, the triglyceride content of the mature fat cells can be obviously increased (p is less than 0.01). And the triglyceride content gradually increased with time, and the triglyceride content of the cells was most significant at the 6 th day of induced differentiation of 3T3-L1 cells, and then the release amount of glycerol was gradually decreased, but still higher than that of the Rosi group. Buckwheat hull flavone can promote lipid decomposition of TG without excessive accumulation of lipid.
4. Influence of buckwheat husk flavone on glycerol release amount of 3T3-L1 preadipocytes
Fig. 8 (c) shows that buckwheat hull flavone can significantly increase the release amount of glycerol from mature adipocytes (p < 0.01) after 2,4, 6, and 8 days of intervention. And the glycerol release amount is gradually increased along with the time, the glycerol release amount of the cells is most obvious on the 6 th day of induced differentiation of the 3T3-L1 cells, and compared with the Rosi group, the glycerol release amount of the PBHE-M4+ Rosi group is respectively increased by 153.99% and 180.07%. The glycerol release was then gradually reduced, but still higher than in the Rosi group. Therefore, the buckwheat hull flavone can activate the lipolysis of lipase while promoting the differentiation of fat cells, and avoid the excessive accumulation of lipid.
Example 7 transcriptomics analysis
As shown in fig. 9, the results of KEGG enrichment analysis indicate that co-incubation of PBHE-M4 and rosiglitazone can regulate multiple signaling pathways, with DEG (differentially expressed genes) enrichment associated with oxidative phosphorylation pathway and thermogenesis pathway being the most significant, explaining lipolysis phenomenon. As shown in FIG. 10 (a, b), based on the analysis of genes related to the difference in expression of Thermogenesis Pathway, 84 DEGs were enriched in the thermogenic Pathway, 70 of which were up-regulated and only 14 were down-regulated. And (3) analyzing genes related to oxidative phosphorylation expression difference. As shown in fig. 10(c, d), a total of 68 were found in the oxidative phosphorylation pathway, of which 65 and 3 were up-and down-regulated, respectively.
Example 8 RT-PCR validation and dynamic evaluation of DEG
The repeatability and reliability of differential gene expression are identified through RNA-seq and computational analysis, and relevant DEGs, including adipogenesis transcription factors, thermogenesis-related genes, uncoupling proteins and selection glucose transporters, are dynamically evaluated at a specific stage of differentiation for qRT-PCR verification of 14 up-regulated genes. Dynamic evaluation showed that synergistic incubation with rosiglitazone and PBHE-M4 significantly upregulated the relative mRNA expression levels of the fat synthesis associated genes containing peroxisome proliferator-activated receptor gamma (Ppar γ), CCAAT enhancer binding protein alpha (C/ebpa). As shown in FIG. 11A, the relative mRNA expression levels of sterol regulatory element-bound transcription factor 1 (Srebp 1 c), glucose transporter 4 (Glut 4) were significantly up-regulated by 5.61-22.14 fold throughout differentiation at day 4, day 6 and day 8. Regarding genes associated with fat browning of 3T3-L1 adipocytes co-cultured with rosiglitazone and PBHE-M4, the relative expression level of Npra was significantly up-regulated on days 2,4 and 6, and the relative expression level of Adrb3, Ucp3, Cox7a1 and Cox8b showed similar trends, showing significant up-regulation on days 4,6 and 8. The relative expression levels of Adcy5 and Ucp2 were significantly up-regulated at day 6 and day 8. Most notably, the level of Fgf21 was significantly upregulated 3.31-4.36 fold relative to expression at all stages of induced differentiation (FIG. 11B). The relative expression of Pka associated with lipid metabolism in adipocytes co-cultured with rosiglitazone and PBHE-M4 was significantly higher than that of rosiglitazone at day 4 and day 6, the relative expression of Atg1 and Mgll was strongly up-regulated by about 6-18 fold. At days 4,6 and 8 of induced differentiation, and even at day 2, Atg1 was activated, demonstrating that lipolysis and adipogenesis were simultaneous (fig. 11C). In conclusion, co-incubation of rosiglitazone and PBHE-M4 could simultaneously strongly up-regulate the relative mRNA expression levels of genes involved in adipogenesis, beige browning and lipolysis at day 6 of induced differentiation, which is highly consistent therewith. Results obtained from RNA-seq.
Claims (7)
1. A preadipocyte browning induction kit, comprising: buckwheat hull flavone.
2. The preadipocyte browning induction kit according to claim 1, wherein: the preparation method of the buckwheat hull flavone comprises the following steps:
1) drying testa Fagopyri Esculenti, pulverizing, adding water, and extracting at high temperature under high pressure;
2) filtering, purifying the filtrate with macroporous resin, concentrating, and lyophilizing to obtain buckwheat hull flavone.
3. The preadipocyte browning induction kit according to claim 2, wherein: the macroporous resin is D101 and AB-8 type macroporous resin.
4. A method for inducing browning of preadipocytes, comprising:
1) inoculating preadipocytes at 37 deg.C with 5% CO2Culturing under the condition until preadipocytes are completely confluent, and continuously inhibiting for 2 days;
2) sucking out the cultured cells in the step 1), and adding buckwheat hull flavone into the differentiation culture mediumCulturing for 2 days, adding buckwheat husk flavone into differentiation culture medium
Culturing for 2 days in medium culture medium, and culturing the buckwheat husk flavone in differentiation culture medium
And
the final concentration of (5) is 10 ~ 100 mug/mL;
3) then, continuously culturing by using a complete culture medium containing 10% FBS, replacing the culture medium every 2 days until 90% of cells show adipocyte phenotype, namely when lipid drops accumulate, the cells are completely differentiated, and finishing the culture;
the components of the culture medium are respectively as follows:
1) complete culture medium 40 ~ 50mL DMEM, 3 ~ 10mL FBS, 450 ~ 550mL penicillin-streptomycin mixed solution;
2) differentiation medium
DMEM 48.8 ~ 49.2.2 mL, 0.5 mM 3-isobutyl-1-methyl xanthine 450 ~ 550 muL, 10 mug/mL insulin 45 ~ 55 muL and 1 muM dexamethasone 45 ~ 55 muL containing 10% FBS and 1% double-antibody respectively in volume fraction, and adding DMEM to supplement the volume fraction to 50 mL;
3) differentiation mediumDMEM 48.8 ~ 49.2.2 mL containing 10% FBS and 1% double-antibody respectively, and 150 ~ 250 mu L of 10 mu g/mL insulin is added to make up to 50 mL.
5. The method of claim 4, wherein the method comprises the steps of: the preadipocytes are 3T3-L1 preadipocytes.
6. The method of claim 5, wherein the method comprises the steps of: the buckwheat hull flavone is in a differentiation culture medium
And
the final concentration of (5) was 10 ~ 80 μ g/mL.
7. The method of claim 6, wherein the method comprises: the buckwheat hull flavone is in a differentiation culture medium
And
the final concentration in (a) is 50 mug/mL.
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| CN117904036A (en) * | 2024-03-19 | 2024-04-19 | 山东第一医科大学(山东省医学科学院) | Method for producing snake oil by culturing fat cells of snake |
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