Application of MLCT in preparation of medicine for preventing and / or treating non-alcoholic fatty liver disease

Abstract

The invention provides application of MLCT in preparation of a medicine for preventing and / or treating non-alcoholic fatty liver disease, and belongs to the technical field of structural lipid application. Researches find that MLCT directly acts on HepG2 cells, and the content of TG and TC in the HepG2 cells can be remarkably reduced; the MLCT acts on the oleic acid-induced HepG2 cells, so that the contents of TG, TC and LDL-C in the oleic acid-induced HepG2 cells can be reduced, the content of HDL-C can be increased, the lipid metabolism of the HepG2 cells can be improved under a certain concentration, the fat synthase can be reduced, and the expression of lipolytic enzyme genes can be improved. The MLCT can weaken the disorder of lipid metabolism by regulating glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine and tyrosine metabolism and the like, and has important significance in preventing the non-alcoholic fatty liver disease and improving triglyceride metabolism.

Description

Technical Field

[0001] The invention belongs to the technical field of structural lipid application, and particularly relates to the application of MLCT in preparing a medicine for preventing and / or treating non-alcoholic fatty liver disease. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is a series of chronic diseases that affect health. It is caused by excessive accumulation of free fatty acids or triglycerides in the liver and is the main cause of hepatocellular carcinoma and chronic liver disease. In addition, NAFLD is associated with a higher risk of diabetes, extrahepatic malignancies, cardiovascular disease, and new-onset heart failure. Generally speaking, the main causes of non-alcoholic fatty liver disease are unbalanced diet (excessive fat intake) and lack of exercise. Therefore, in addition to exercise, the development of NAFLD can be controlled through dietary intervention.

[0003] MLCT is a lipid with medium-chain and long-chain fatty acids randomly positioned on the same glycerol backbone. It is metabolized in two different ways: long-chain triglycerides (LCTs) and medium-chain triglycerides (MCTs). The unique digestion and absorption method of MLCT not only supplements long-chain essential fatty acids, but also avoids the harm of excessive MCT concentration to the human body, maintaining the advantages of MCT in terms of easy digestion and providing a quick energy source. Compared with LCT, MCT, and physical mixed oils of LCT and MCT, the medium-chain and long-chain fatty acids released by MLCT are hydrolyzed faster and more gently than MCT and LCT, releasing medium-chain fatty acids that enter the portal vein system to quickly provide energy, while long-chain fatty acids and monoglycerides enter the lymphatic system and are carried into the blood to the tissues. At present, the preventive effect of MLCT on fat accumulation or NAFLD has not been described in human liver cancer cells. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the purpose of the present invention is to provide the use of MLCT in the preparation of drugs for preventing and / or treating non-alcoholic fatty liver disease and improving triglyceride metabolism.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides the use of MLCT in preparing a medicine for preventing and / or treating non-alcoholic fatty liver disease. The MLCT is synthesized from olive oil and medium-chain triglycerides.

[0007] The present invention also provides the use of MLCT in preparing a medicine for preventing and / or treating lipid metabolism disorders, wherein the MLCT is synthesized from olive oil and medium-chain triglycerides.

[0008] The present invention also provides the use of MLCT in preparing a medicine for improving lipid metabolism disorder of HepG2 cells, wherein the MLCT is synthesized from olive oil and medium-chain triglycerides.

[0009] Preferably, the MLCT downregulates the expression of FAS, ACC, SREBP-1c and PPARγ mRNA.

[0010] Preferably, the MLCT upregulates the expression of ATGL and CPT1A mRNA.

[0011] Preferably, the MLCT reduces the TG and TC contents in cells.

[0012] Preferably, the MLCT regulates glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine and tyrosine metabolism.

[0013] Preferably, in the MLCT, the content of medium-chain triglycerides is 70-85%, the distribution of long-chain fatty acids at the Sn-2 position is 80-82%, and the content of oleic acid at the Sn-2 position is 40-50%.

[0014] Preferably, the preparation method of MLCT comprises the following steps: mixing olive oil and medium-chain triglycerides, adding immobilized lipase, reacting to obtain a crude product, and separating and purifying the crude product to obtain MLCT; the immobilized lipase is LipozymeTLIM.

[0015] The present invention also provides a medicine for preventing and / or treating lipid metabolism disorder, wherein the medicine uses the MLCT prepared by the above preparation method as an active ingredient.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention found that MLCT can significantly reduce the TG and TC contents in HepG2 cells by directly acting on HepG2 cells; MLCT can reduce the TG, TC and LDL-C contents in oleic acid-induced HepG2 cells, increase the HDL-C content, improve the lipid metabolism of HepG2 cells at a certain concentration, reduce the expression of fat synthase and increase the expression of lipase genes. MLCT can reduce the disorder of lipid metabolism by regulating glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine and tyrosine metabolism, etc., and is of great significance for preventing non-alcoholic fatty liver, preventing obesity and improving triglyceride metabolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Effects of different concentrations of triglyceride sample oil on HepG2 cell viability;

[0019] Figure 2 :The effects of different concentrations of triglyceride sample oil on total triglyceride and total cholesterol in HepG2 cells;

[0020] Figure 3 :Effect of oleic acid addition on HepG2 cell viability;

[0021] Figure 4 :Oleic acid induces adipogenesis in HepG2 cells (40×);

[0022] Figure 5 :Effects of different concentrations of triglyceride sample oil on total triglycerides, total cholesterol, low-density lipoprotein cholesterol and high-density lipoprotein cholesterol in HepG2 cells induced by oleic acid;

[0023] Figure 6 : Representative micrographs of HepG2 cells stained with Oil Red O (20×);

[0024] Figure 7 :Effects of different concentrations of triglyceride sample oil on fat-related genes in HepG2 cells induced by oleic acid;

[0025] Figure 8 : Representative TIC chromatograms of the control group, model group, and olive oil group;

[0026] Fig. 9 : Representative TIC chromatograms of the physical mixed oil group;

[0027] Fig.10 : Representative TIC chromatograms of the MLCT group;

[0028] Fig.11 : Metabolite classification pie chart;

[0029] Fig.12 : PCA analysis results of total samples in different groups;

[0030] Fig.13 : PLS-DA score scatter plot and ranking validation diagram between different groups;

[0031] Fig.14 : Volcano plot of differential metabolites among different groups;

[0032] Fig.15 : KEGG classification diagram of differential metabolites among different groups. DETAILED DESCRIPTION

[0033] The present invention provides the use of MLCT in preparing a medicine for preventing and / or treating non-alcoholic fatty liver disease, in preparing a medicine for preventing and / or treating lipid metabolism disorder, and in preparing a medicine for improving lipid metabolism disorder in HepG2 cells.

[0034] The present invention studies and explores the effect of MLCT directly acting on HepG2 cells on lipid accumulation and the effect of MLCT on lipid accumulation of human liver cancer cells induced by oleic acid. The experimental results show that compared with the control group, MLCT significantly reduced the TG and TC contents in HepG2 cells. Compared with the model group, MLCT significantly reduced the TG, TC, and LDL-C contents in oleic acid-induced HepG2 cells, and increased the HDL-C content; compared with the model group, the MLCT intervention group can down-regulate the expression of FAS, ACC, SREBP-1cmRNA, and PPARγmRNA at a certain concentration (P<0.05), and up-regulate the expression of ATGL and CPT1AmRNA in cells (P<0.05). MLCT can improve HepG2 cell lipid metabolism at a certain concentration, reduce fat synthase, and increase the expression of lipase genes. MLCT can reduce lipid metabolism disorders by regulating glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine and tyrosine metabolism, etc. MLCT is of great significance for preventing non-alcoholic fatty liver, preventing obesity, and improving triglyceride metabolism. The effective concentration of MLCT of the present invention is 50-100 μg / mL.

[0035] The MLCT of the present invention is synthesized from olive oil and medium-chain triglycerides. Preferably, in the MLCT, the content of medium-chain triglycerides is 70-85%, the distribution of long-chain fatty acids at the Sn-2 position is 80-82%, and the content of oleic acid at the Sn-2 position is 40-50%. The preparation method of the MLCT comprises the following steps: olive oil and medium-chain triglycerides are mixed, immobilized lipase is added, a crude product is obtained by reaction, and the crude product is separated and purified to obtain MLCT; the immobilized lipase is LipozymeTLIM.

[0036] As an optional embodiment, the present invention mixes olive oil and medium-chain triglycerides in a mass ratio of 3:2 to 7:3, and adds 6 to 7.5 wt% (based on the total mass of olive oil and medium-chain triglycerides) of LipozymeTLIM to the mixed system, and performs an ester exchange reaction at a temperature of 59 to 60°C for 2 to 4 hours to obtain a crude product. The present invention purifies and separates the crude product obtained by the reaction: the crude product is mixed with anhydrous ethanol, stirred at 35 to 37°C for 25 to 35 minutes, and the ethanol phase is discarded; the product is mixed with silica gel, stirred and centrifuged, and the precipitate is discarded; n-hexane is added to the supernatant for extraction; the product obtained after the extract is evaporated under reduced pressure is the purified MLCT.

[0037] In the preparation of MLCT of the present invention, the olive oil is preferably virgin olive oil; the fatty acids in the olive oil are mainly oleic acid and linoleic acid; the oleic acid content is 75-80%, and the linoleic acid content is 5-7%. The medium-chain triglycerides are mainly caprylic acid and capric acid, the caprylic acid content is 60-62%, and the capric acid content is 38-40%.

[0038] The present invention also provides a drug for preventing and / or treating lipid metabolism disorders, wherein the drug comprises MLCT prepared by the above-mentioned preparation method as an active ingredient. The content of the active ingredient in the drug of the present invention is 1 to 99.99 wt%, preferably 50 to 90 wt%. Preferably, the drug of the present invention further comprises a pharmaceutically acceptable carrier, and a pharmaceutically acceptable pharmaceutical excipient is added to the drug to ensure that the prepared drug for preventing and / or treating lipid metabolism disorders is convenient for preparation and practical application.

[0039] In the following examples, unless otherwise specified, all methods are conventional. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0040] In a specific embodiment of the present invention, human liver cancer cell HepG2 was purchased from the Chinese Academy of Sciences (Shanghai, China); MEM culture medium was purchased from Pronocell Life Science (Wuhan) Co., Ltd.; CCK-8 kit was purchased from Lanjieke Technology (Hefei) Co., Ltd.; TP assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; TG assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; T-CHO assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; HDL-C assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; LDL-C assay kit was purchased from Nanjing Jiancheng Bioengineering Institute; Oil Red O staining kit was purchased from Beyotime Biotechnology (Shanghai) Co., Ltd.; oleic acid was purchased from MedChemexpress Biotechnology, USA.

[0041] In a specific embodiment of the present invention, olive oil was purchased from Yihai Kerry Grain and Oil Industry (Wuhan) Co., Ltd., and the olive oil contained 79.41% oleic acid, 6.59% linoleic acid, and 14% other fatty acids; medium-chain triglycerides (MCT) were purchased from McLean Biochemical Technology Co., Ltd. (Shanghai) Co., Ltd., with a purity of ≥98%, and MCT contained 61.33% caprylic acid and 38.67% capric acid.

[0042] Preparation method of physical mixed oil: olive oil and medium chain triglycerides are mixed in a mass ratio of 3.15:1.85;

[0043] Preparation method of MLCT: 5g of olive oil and MCT were placed in a reaction vessel at a mass ratio of 3.15:1.85, and 7wt% of immobilized lipase (Lipozyme TLIM) was added. The mixture was stirred in a water bath at 60°C for 3h under light protection. After the reaction, the reaction system was centrifuged at 4200rpm for 5min, and the supernatant was the reaction product (crude product). Separation and purification: The crude product was mixed with anhydrous ethanol at a ratio of 1:1 (V / V), stirred with a magnetic stirrer for 30min at 36°C, the ethanol phase was discarded, and the extraction was repeated 6 times for deacidification. 30g of the deacidified sample was mixed with 1.8g of silica gel, stirred with a magnetic stirrer for 2h at room temperature, and centrifuged at 4200rpm for 5min. The supernatant was extracted with n-hexane at a volume ratio of 1:2 and extracted twice. The extract was placed in a brown bottle, left to stand at -20°C overnight, and filtered with filter paper the next day. The n-hexane was removed by vacuum evaporation at 70°C and 100 rpm using a rotary evaporator. The purified sample was frozen at -20°C. The speed of the magnetic stirrer was 500 rpm and the power was 16W.

[0044] Example 1

[0045] 1. Cell culture

[0046] HepG2 cells were collected at 1×10 6 Inoculate each cell in a T25 culture flask, add 6 mL of MEM medium containing 10% fetal bovine serum (FBS) and 1% P / S, and culture in an incubator (37°C, 5% CO2). When the cell confluence reaches more than 80%, subculture.

[0047] 2. Cell passaging

[0048] Discard the culture medium in the T25 culture flask, add 2mL of trypsin to digest for 2-3 minutes, then add 2mL of MEM cell culture medium to terminate digestion, blow the culture medium with a pipette to make the cells fall off from the bottom of the culture flask and disperse in the culture medium. Centrifuge the blown-off cell suspension at 1000rpm for 5 minutes, discard the supernatant, add 12mL of MEM cell culture medium to resuspend the cells by blowing, and divide into two T25 culture flasks.

[0049] 3. Cell cryopreservation and recovery

[0050] After trypsin digestion, blowing off, centrifugation and removal of supernatant, the precipitated cells were collected (10 6 ~10 7 / mL), add 1mL of serum-free cell freezing solution and dispense into 1mL cryotubes. Quickly take out the cryotube from the liquid nitrogen tank, place it in a 37℃ water bath to shake and melt, and quickly add it to a 15mL centrifuge tube containing 5mL of MEM cell culture medium, mix it, and centrifuge it at 1000rpm for 5min; discard the supernatant, resuspend the cell pellet with 1mL of culture medium, transfer it to a T25 culture flask, add 5mL of culture medium, mix it, and transfer it to a 5% CO2 incubator for culture and static.

[0051] 4. Cell Viability Assay

[0052] Cell viability was measured using Cell Counting Kit-8 (CCK-8).

[0053] HepG2 cells were cultured at 1×10 4 Each well was inoculated in a 96-well plate and placed in an incubator (37°C, 5% CO2) for culture. After 1 hour of culture, the original culture medium was discarded and cultured for 24 hours with a culture medium containing 0μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 300μg / mL, 400μg / mL and 500μg / mL of different triglyceride sample oils (olive oil, physical mixed oil, MLCT). Then 10μL of CCK-8 solution was added to each well and incubated at 37°C for another 2 hours to calculate the cell viability.

[0054] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the cytotoxicity of different triglyceride samples on HepG2 cells is dose-dependent. At low concentrations (<300μg / mL), there is no significant effect on cell viability (P>0.05, cell viability>95%). At a concentration of 300μg / mL, olive oil and physical mixed oil significantly reduced HepG2 cell viability (P<0.05), and the cell viability of the MLCT group was also less than 95%. With the increase of triglyceride sample oil concentration, the inhibitory effect on cell growth increased (cell viability <95%). The results show that high-concentration triglyceride samples (≥300μg / mL) have toxic effects on HepG2 cells. Triglyceride sample concentrations of 25μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL were selected for the next step of the experiment.

[0055] 5. Triglyceride sample intervention HepG2 cell experiment

[0056] HepG2 cells were seeded into 6-well plates and incubated with different triglyceride samples (olive oil, physical mixed oil, MLCT) at concentrations of 0μg / mL, 25μg / mL, 50μg / mL, 100μg / mL, and 200μg / mL for 24h. The total triglyceride (t-TG) and total cholesterol (TC) contents were measured using detection kits (TG and T-CHO).

[0057] Results Figure 2 , Figure 2 In the figure, a is the total triglyceride content in HepG2 cells, b is the total cholesterol content in HepG2 cells, and the bars show the mean ± standard deviation of the samples under each condition. Different lowercase letters indicate significant differences (P<0.05). The results showed that for the total triglyceride content, compared with the control group, with the increase of olive oil, physical mixed oil and MLCT concentration (25-100μg / mL), the t-TG content tended to decrease, and the differences among the three were not very large. Among them, compared with the olive oil and MLCT groups, the t-TG content of the mixed oil was lower at a concentration of 25-100μg / mL. For the total cholesterol content, compared with the control group, the TC content of olive oil at a concentration of 25-100μg / mL tended to decrease. The physical mixed oil group and the MLCT group had the lowest TC content at concentrations of 25μg / mL and 50μg / mL, both of which were lower than the olive oil group. This shows that physical mixed oil and MLCT are rich in medium-chain fatty acids and can reduce the content of TC.

[0058] 6. Triglyceride samples intervene in oleic acid-induced adipogenesis in HepG2 cells

[0059] (1) Oleic acid induces adipogenesis in HepG2 cells

[0060] The concentration of oleic acid (non-cytotoxic) was screened by CCK-8 method: HepG2 cells (logarithmic growth phase) in T25 culture flasks were digested and 1×10 4 HepG2 cells were grown to 80-90% and different concentrations of oleic acid (0.1mM, 0.2mM, 0.3mM, 0.4mM, 0.5mM, 0.6mM, 0.7mM) were added for 24h. Figure 3 shown.

[0061] The results showed that after 24 hours of oleic acid treatment, when the oleic acid concentration was between 0.1 and 0.3 mM, the fat accumulation of HepG2 cells increased in a dose-dependent manner. When the oleic acid concentration reached 0.4 mM, the cell activity tended to decrease. When the oleic acid concentration reached 0.6 to 0.7 mM, the toxicity was higher (cell survival rate <95%).

[0062] Observation of the optimal OA-induced lipid droplet formation (Oil Red O staining): HepG2 cells were seeded in 6-well plates (6×10 5 / well), and after culturing for 24 hours, the culture medium was aspirated and divided into blank control group and model group. The model group and control group used complete culture medium and cultured for 24 hours. Oleic acid with a final concentration of 0.3mM was added to the complete culture medium of the model group. The culture medium was aspirated and washed once with PBS (1mL), fixed with 4% paraformaldehyde (1mL) at room temperature for 30min, washed again with PBS (1mL), added with Oil Red O working solution, incubated at room temperature for 30min, discarded the staining solution, and washed once and twice with staining washing solution and PBS respectively. The lipid droplets were observed with an inverted microscope and recorded. The results of Oil Red staining of the control group and model group are shown in the figure. Figure 4 The results showed that more lipid droplets were formed in the model group cells due to OA induction, indicating that the model was successfully established.

[0063] (2) Triglyceride sample intervention in oleic acid-induced HepG2 cell experiment

[0064] The experiment was divided into a control group, a model group, and a triglyceride sample (olive oil, physical mixed oil, MLCT) intervention group. Each group was repeated three times. The specific experimental operation was as follows: 6×10 5 HepG2 cells were cultured in the presence of 0.3 mM oleic acid in the model group and the intervention group of different triglyceride samples. After the cell growth reached about 70% to 80%, the different triglyceride sample intervention groups were added with culture medium containing sample oil (the concentration of triglyceride sample added in each group was 25 μg / mL, 50 μg / mL, 100 μg / mL and 200 μg / mL, respectively) and cultured under experimental conditions for 24 hours. The model group and the control group were cultured with complete culture medium for 24 hours. The culture medium was discarded, and the model group and the different triglyceride sample intervention groups were replaced with culture medium containing 0.3 mM oleic acid in an incubator (37°C, 5% CO2) for 24 hours to induce HepG2 cell adipogenesis. The total triglyceride (t-TG) and total cholesterol (TC) contents were measured using detection kits (TG and T-CHO), respectively. The protein concentration was determined using a BCA protein assay kit.

[0065] Results Figure 5 , Figure 5 In the figure, a is the total triglyceride content in HepG2 cells, b is the total cholesterol content in HepG2 cells, c is the high-density lipoprotein cholesterol content in HepG2 cells, and d is the low-density lipoprotein cholesterol content in HepG2 cells. The bars show the mean ± standard deviation of the samples under each condition. Different lowercase letters indicate significant differences (P<0.05).

[0066] Figure 5Figure a shows that the intracellular t-TG content in the model group increased, about 4.13 times that of the control group, and the fatty liver cell model was successfully established. The intracellular t-TG content tended to decrease with the increase of MLCT concentration. The intracellular t-TG content in the MLCT intervention group was significantly lower than that in the model group (P<0.05). The t-TG content in the MLCT intervention group (50, 100 and 200 μg / mL) decreased significantly. With the increase of the concentration of the olive oil intervention group, the intracellular t-TG content tended to increase first and then decrease. Among them, the intracellular t-TG content in the 50 μg / mL olive oil intervention group reached about 0.4 mM / g protein, which was slightly lower than the modeling intervention group. At a certain concentration, olive oil will increase intracellular free fatty acids and weaken the lipid-lowering effect. With the increase of the concentration of the physical mixed oil intervention group, the intracellular t-TG content tended to decrease, but it was higher than the MLCT intervention group.

[0067] Figure 5 Figure b shows that the model group can significantly increase the TC content in HepG2 cells compared with the control group (P<0.05). The intracellular TC content in the modeling intervention group increased, which was about 2.45 times that of the normal control group. Compared with the modeling intervention group, the TC content in the 25μg / mL olive oil intervention group, the 100μg / mL physical mixed oil and the MLCT intervention group decreased significantly (P<0.05). Among them, MLCT concentrations of 50μg / mL and 100μg / mL can more effectively reduce the intracellular TC content.

[0068] Figure 5 Figure c shows that the LDL-C content in HepG2 cells of the model group was significantly increased compared with the control group (P<0.05). The LDL-C content in the cells of the triglyceride sample intervention group tended to decrease compared with the model group. When the oil concentration was 25μg / mL, the LDL-C content was in the order of MLCT>physical mixed oil>olive oil from high to low. When the oil concentration was 50μg / mL, the LDL-C content was in the order of olive oil>physical mixed oil>MLCT from high to low. When the oil concentration was 100μg / mL, the LDL-C content of the three was not much different. When the oil concentration was 200μg / mL, the LDL-C content was in the order of MLCT>olive oil>physical mixed oil from high to low. Compared with the model group, olive oil and physical mixed oil, MLCT had the lowest LDL-C content at a concentration of 50μg / mL (P<0.05).

[0069] Figure 5Figure d shows that the HDL-C content in the model group cells was reduced compared with the control group. After incubation with different triglyceride sample oils, the accumulation of HDL-C in the cells tended to increase compared with the model group. At different concentrations (25-200 μg / mL), the increase in HDL-C in the cells between the groups was not much different. When the oil concentration was 200 μg / ml, the HDL-C content in the MLCT group was the highest. The results showed that MLCT can inhibit the abnormalities of LDL-C and HDL-C in HepG2 cells caused by oleic acid, and the effect is better than olive oil and physical mixed oil, so that the relative stability of cholesterol is maintained.

[0070] Oil Red O staining was used to observe the lipid droplet formation in each group: the staining steps were the same as step (1).

[0071] Oil red staining results Figure 6 As shown, Figure 6 In the figure, a is the control group and the model group, b is the olive oil group, c is the physical mixed oil group, and d is the MLCT group. The results showed that the oil red staining results of cells in each group were similar to those in the control group and the model group. Figure 5 The t-TG levels detected in the cells were basically consistent.

[0072] 7. Effects of MLCT on genes related to lipogenesis / decomposition in HepG2 cells

[0073] (1) Total RNA extraction

[0074] Sterilize 1.5mL EP tubes, 200μL PCR tubes, 1000μL, 200μL and 10μL pipette tips one day in advance. Prepare DEPC water: In a fume hood, dissolve 50μL in 50mL ultrapure water and let stand at 37℃ for 2h. Sterilize at 121℃ for 20min, cool to room temperature after sterilization, and store 1mL per tube at -20℃.

[0075] 6×10 cells were seeded in each well of a six-well plate. 5 HepG2 cells were cultured for 24 hours under experimental conditions after the cell growth reached about 60% to 70%. Different triglyceride sample oils (olive oil, physical mixed oil, MLCT) intervention groups (the concentration of triglyceride samples added in each group was 50μg / mL and 100μg / mL, respectively), and the normal control group and model group were cultured in complete medium for 24 hours. The original medium was discarded, and the model group and different triglyceride sample oil intervention groups were replaced with medium containing 0.3mM oleic acid for 24 hours for modeling.

[0076] Aspirate the culture medium, add pre-cooled PBS to rinse, discard PBS, place the six-well plate on a flat ice pack, add 550μL Trizol reagent, blow up and down to lyse the cells, then transfer the cells to a 1.5mL EP tube, let it stand at 4℃ for 10min, add 100μL chloroform, turn it upside down for 15s, let it stand on ice for 10min, centrifuge (4℃, 12000rpm, 15min), transfer the upper aqueous phase to a new EP tube, add 500μL isopropanol to the EP tube, turn it upside down for 15s, let it stand on ice for 10min, after the standing period, centrifuge at 4℃, 12000rpm for 15min, aspirate the supernatant to keep the RNA precipitate, add 1mL 75% ethanol solution, wash the RNA, centrifuge at 4℃, 12000rpm for 5min, discard ethanol, and then follow the above operation and wash it again with ethanol solution. Let it stand on ice to dry for 10min to evaporate the residual ethanol. Finally, add an appropriate amount of DEPC water to dissolve the RNA precipitate, pipet up and down to dissolve the RNA, vortex and shake for 5 to 10 seconds, and then test the RNA concentration. It can be used for cDNA synthesis or stored in a -80°C refrigerator.

[0077] (2) RNA concentration detection and cDNA synthesis

[0078] RNA denaturation: After the reaction mixture was briefly centrifuged, the RNA solution was incubated in a water bath at 37°C for 15 min, then in a water bath at 60°C for 10 min, and then immediately cooled on ice.

[0079] RNA concentration detection: The RNA sample concentration was detected by ultra-micro UV-visible photometer. The 260 / 280nm ratio was used to characterize the nucleic acid concentration. The ratio of high-quality RNA samples was generally 1.80-2.0, and the ratio of high-quality DNA samples was 1.60-1.80. Take 500ng RNA and use the reverse transcription kit provided by Novozyme Biotech Co., Ltd. to reverse transcribe and synthesize cDNA.

[0080] cDNA synthesis: prepare the reverse transcription system according to Table 1. Reverse RNA to cDNA under the conditions of 37°C for 15 min, 98°C for 5 min, and 4°C for 30 min in a water bath, and store the cDNA at -20°C.

[0081] Table 1 Reverse transcription reaction system

[0082] Reagent name Dosage All-In-One 5×RTMasterMix 4μL Total RNA or poly(A)+mRNA Variable (1ng-2μg / rxn) <![CDATA[Nuclease-freeH2O]]> Make up to 20 μL

[0083] The cDNA housekeeping gene (primer) and other gene primers were set aside (melted). The housekeeping primer and other primers were 1×. The RT-qPCR related gene primers were designed as shown in Table 2.

[0084] Table 2 Gene primer sequence list

[0085]

[0086]

[0087] The PCR reaction system was 10 μL, and the amount of each component added is shown in Table 3. The target gene GAPDH was used as the internal reference gene, and the control group cells were used as the control. The cycle number Ct value determined by RT-qPCR (Bio-Rad CFX96 fluorescence quantitative PCR instrument) was used to calculate the relative expression of its mRNA (2 -ΔΔCt ).

[0088] Table 3 RT-qPCR reaction system

[0089] Reagents Amount per tube cDNA 1μL Forwardprime working fluid 0.4μL Reverseprimer working solution 0.4μL SYBR Green qPCR Master Mix 5μL Add RNase-free water to 3.2μL

[0090] The results are as follows Figure 7 shown. Figure 7 In the figure, a is the relative expression level of FAS mRNA, b is the relative expression level of ACC mRNA, c is the relative expression level of SREBP-1c mRNA, d is the relative expression level of PPARγ mRNA, e is the relative expression level of ATGL mRNA, and f is the relative expression level of CPT1A mRNA. Figure 7 Figures a and b show that the expression of ACC and FAS mRNA in the model group was higher than that in the normal control group, and the expression of FAS mRNA in the model group increased significantly (P<0.05). This indicates that lipogenesis-related enzymes are activated by excessive free fatty acids, thereby accelerating the synthesis of triglycerides in hepatocytes. Compared with the model group, different concentrations of olive oil, physical mixed oil and MLCT can downregulate the expression of FAS and ACC genes to a certain extent. Among them, olive oil downregulated the expression of FAS and ACC genes more significantly at 50μg / mL (P<0.05). Compared with the physical mixed oil, MLCT downregulated the expression of ACC gene more significantly at 50μg / mL (P<0.05). Compared with the olive oil group, MLCT downregulated the expression of ACC gene more significantly at 100μg / mL (P<0.05). Reducing the expression of ACC and FAS genes can reduce fat synthesis, inhibit ACC expression, promote CPT-1 activity, and increase mitochondrial β-oxidation. The results showed that MLCT could affect mitochondrial lipolysis in hepatocytes and reduce initial lipid synthesis by downregulating the expression of enzymes involved in lipolysis.

[0091] Figure 7Figures c and d show that compared with the control group, the expression of SREBP-1c gene in the model group increased. Compared with the model group, different concentrations of olive oil, physical mixed oil and MLCT could downregulate the expression of SREBP-1c gene. At a concentration of 50 μg / mL, compared with the olive oil intervention group and the physical mixed oil intervention group, the MLCT intervention group was more effective in downregulating the expression of SREBP-1c mRNA in cells. Compared with the control group, the expression of PPARγ mRNA in the model group was significantly increased (P<0.05). Compared with the model group, different concentrations of olive oil, physical mixed oil and MLCT could downregulate the expression of PPARγ mRNA to a certain extent. At a concentration of 50 μg / mL, compared with the olive oil intervention group and the physical mixed oil intervention group, the MLCT intervention group was more effective in downregulating the expression of PPARγ mRNA in cells. The results showed that MLCT played a lipid-lowering role in vitro, downregulated the expression of adipogenesis-related genes, and reduced lipid synthesis and uptake in HepG2 cells.

[0092] Figure 7 Figures e and f show that the expression of ATGLmRNA and CPT1AmRNA in the model group cells was significantly lower than that in the normal control group (P<0.05). Compared with the model group, olive oil, physical mixed oil and MLCT intervention upregulated the expression of ATGLmRNA in cells. Compared with the model group, after MLCT intervention, the expression of CPT1A gene in cells was significantly upregulated, and the upregulation trend became more obvious with the increase of concentration (50μg / mL, 100μg / mL). The results show that MLCT can upregulate the expression of genes related to lipolysis and improve lipid accumulation in HepG2 cells.

[0093] 8. Metabolomics-based study on the effect of MLCT on oleic acid-induced HepG2 cells

[0094] (1) HepG2 cell grouping intervention

[0095] The experiment was divided into a control group, a model group, and a triglyceride sample (olive oil, physical mixed oil, MLCT) intervention group. Each group was repeated three times. The specific experimental operation was as follows: T75 culture flasks were inoculated with 4.6875×10 6 HepG2 cells were grown for 24 hours, and the different triglyceride sample intervention groups were added with culture medium containing sample oil (concentration of 100 μg / mL) and cultured under experimental conditions for 24 hours, and the model group and the control group were cultured with complete culture medium for 24 hours. The culture medium was discarded, and the model group and the different triglyceride sample intervention groups were replaced with culture medium containing 0.3 mM oleic acid and cultured in an incubator for 24 hours to induce HepG2 cells to become adipogenic.

[0096] (2) Determination of HepG2 cell metabolomics after intervention

[0097] Collect cells: After cell culture, remove the culture medium, wash twice with PBS (pre-cooled), discard the supernatant, and after trypsin digestion, terminate the trypsin action with pre-cooled PBS solution and wash the cells (twice). Transfer the cells into a centrifuge tube and centrifuge at 1000g for 1 min at 4°C. The cell pellet is placed in a 2mL cryotube, quickly frozen in liquid nitrogen for 15 minutes, and then stored at -80°C for subsequent determination.

[0098] Extraction of cell metabolites: The cell sample was taken out of the -80℃ freezer, and a methanol (80%) aqueous solution (300μL) was added, and the sample was quickly frozen (liquid nitrogen) for 5min, and then thawed on ice and vortexed for 30s, ultrasonicated for 6min, and finally centrifuged at 4℃, 5000rpm, for 1min. The supernatant was freeze-dried and added with a methanol (10%) solution of the same volume as the sample, and finally detected and analyzed by LC-MS. Quality control (QC) sample: An equal volume of experimental sample was taken and mixed. Blank sample: Methanol (53%) aqueous solution, the same pretreatment process as the experimental sample was performed.

[0099] Representative TIC chromatograms of each group of experimental samples are shown in Figure 8 to Figure 10 (+: positive ion mode; -: negative ion mode). Figure 8 In the figure, a is the control group, b is the model group, and c is the olive oil group; Fig. 9 It is a physical mixed oil group; Fig.10 The results showed that there were some differences in the TIC profiles among the control group, model group, and intervention groups with different triglyceride samples (olive oil, physical mixed oil, and MLCT), indicating that metabolites underwent certain changes under different intervention conditions.

[0100] Metabolite detection: Vanquish UHPLC chromatograph, chromatographic column (C18 Hypersil Gold column), flow rate 0.2 mL / min, column temperature 40°C. Mobile phase A and B were 0.1% formic acid and methanol, respectively. Q Exactive TM The HF mass spectrometer was operated in a scan range of 100 to 1500 m / z, a spray voltage of 3.5 kV, an ion introduction RF level of 60, an auxiliary gas flow rate of 10 L / min, a sheath gas flow rate of 35 psi, an ion transfer tube temperature and an auxiliary gas heater temperature of 320°C and 350°C, respectively, in positive / negative ion mode, and MS / MS secondary scanning.

[0101] The results are as follows Fig.11As shown in the figure, the metabolites in HepG2 cells after treatment with different triglyceride samples (olive oil, physical mixed oil, MLCT) in the total ion (positive and negative ion) mode were chemically classified and statistically analyzed, and 795 metabolites were found, including lipids and lipid molecules (282), organic acids and their derivatives (183), organic heterocyclic compounds (104), nucleosides, nucleotides and analogues (90), organic oxides (48), benzene ring-type (48), phenylpropanoids and polyketides (21), organic nitrogen compounds (15), and alkaloids and their derivatives (4).

[0102] Metabolite identification: CD 3.3 (Compound Discoverer 3.0, Thermo Fisher) search software processes the data result (.raw) file, screens parameters such as the mass-to-charge ratio and retention time of each metabolite, uses the first QC to correct the peak area, sets 5ppm mass deviation, 30% signal intensity deviation, adduct ion, minimum signal intensity and other information to extract peaks, then quantifies the peak area, integrates the target ion, and predicts the molecular formula. Peaks are compared with the databases Masslist, mzVault and mzCloud (https: / / www.mzcloud.org / ), background ions are removed with blank samples, and the quantitative results are standardized to identify metabolites. CentOS version 6.6 Linux operating system, Python (version 2.7.6) and R (R-3.4.3) software will complete some data processing.

[0103] Statistical data for analysis: Metabolites were annotated and identified using the KEGG, HMDB, and LIPIDMaps databases. Multivariate statistical analysis: metaX software was used to transform the data, and the VIP value of each metabolite could be obtained using principal component and partial least squares discriminant analysis. Univariate analysis of differential metabolites: The t-test was used to obtain the statistical significance (P value) of metabolites and the difference fold (FC value) between the two groups. VIP>, P value <0.05 and FC≤0.5 or FC≥2 were used as criteria for screening differential metabolites. Bubble charts and volcano charts were drawn by the R package ggplot2. Cluster heat maps were drawn by the R package Pheatmap. Metabolite data were normalized using z-score. Metabolite metabolic pathways and functions were studied using the KEGG database: metabolic pathway enrichment (x / n>y / n); metabolic pathways were significantly enriched (P<0.05).

[0104] The PCA analysis results of the total sample are as follows Fig.12As shown. The results showed that multiple metabolites coexisted in the model group, olive oil group and mixed oil group, and the difference between the groups was relatively small. The control group and MLCT group had relatively large differences compared with the model group, olive oil group and mixed oil group. Especially along the first principal component PC1 (PC1 and PC2 accounted for 38.87% and 13.33% of the total principal components, respectively).

[0105] The PLSDA method was used to reanalyze the cell metabolomics data of each treatment group in pairs, and 200 random perturbations were performed to identify the markers of each sample, followed by modeling and prediction, and two regression lines with R2 and Q2 values ​​were obtained. Fig.13 shown. Fig.13 In total ion mode: a represents the control group and the model group; b represents the control group and the olive oil group; c represents the control group and the physical mixed oil group; d represents the control group and the MLCT group; e represents the model group and the olive oil group; f represents the model group and the physical mixed oil group; g represents the model group and the MLCT group; h represents the olive oil group and the MLCT group. The results show that there are obvious differences between the cells of each treatment group under the PLSDA analysis method, indicating that different treatment groups have more or less non-negligible effects on the body's metabolism. And the R2 value>Q2 value, and the intercept of the Q2 regression line and the Y axis <0, it can be found that the model is not "overfitting" and can describe the sample well.

[0106] The thresholds were set as VIP>1.0, FC>1.2 or FC<0.833 and P-value<0.05 to screen differential metabolites. The results are as follows Figure 14-15 and Tables 4 to 6. The volcano plots of the differential metabolites between the comparison groups are shown in Fig.14 shown. Fig.14 (Total ion mode): a represents the olive oil group and the control group; b represents the olive oil group and the model group; c represents the physical mixed oil group and the control group; d represents the physical mixed oil group and the model group; e represents the model group and the control group; f represents the MLCT group and the control group; g represents the MLCT group and the model group; h represents the MLCT group and the olive oil group. KEGG classification diagram of differential metabolites is shown in Figure 2. Fig.15 As shown, Fig.15 (under total ion mode): a represents the model group and the control group; b represents the olive oil group and the control group; c represents the physical mixed oil group and the control group; d represents the MLCT group and the control group; e represents the olive oil group and the model group; f represents the physical mixed oil group and the model group; g represents the MLCT group and the model group; h represents the MLCT group and the olive oil group.

[0107] Fig.14 and Fig.15The results showed that the differential metabolic pathways between the model group and the control group mainly included the nervous system, digestive system, metabolism of cofactors and vitamins, nucleotide metabolism, lipid metabolism, energy metabolism, carbohydrate metabolism, amino acid metabolism, and metabolism of other amino acids. The number of differences in metabolites between the two groups was more in the cofactor and vitamin metabolism pathway, digestive system, nucleotide metabolism pathway, lipid metabolism pathway, amino acid metabolism pathway, and other amino acid metabolism. The number of differences in metabolites between the olive oil group and the control group was more in the amino acid metabolism pathway, lipid metabolism pathway, digestive system, cofactor and vitamin metabolism pathway. The number of differences in metabolites between the physical mixed oil group and the control group was more in the carbohydrate metabolism pathway, digestive system, nucleotide metabolism pathway, metabolism of cofactors and vitamins, lipid metabolism pathway, amino acid metabolism pathway, and carbohydrate metabolism pathway. The number of differences in metabolites between the MLCT group and the control group was more in the lipid metabolism pathway and amino acid metabolism pathway. The number of metabolites with different metabolites between the olive oil group and the model group was more in the lipid metabolism pathway. The number of metabolites with different metabolites between the physical mixed oil group and the model group was more in the digestive system, nucleotide metabolism pathway, and amino acid metabolism pathway. The number of metabolites with different metabolites between the MLCT group and the model group was more in the digestive system, nucleotide metabolism pathway, lipid metabolism pathway, and amino acid metabolism pathway. The number of metabolites with different metabolites between the MLCT group and the olive oil group was more in the nucleotide metabolism pathway and amino acid metabolism pathway.

[0108] Table 4 Differential metabolites in total ion mode caused by model group and control group

[0109]

[0110] Table 5 Differential metabolites in total ion mode caused by MLCT group & model group

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[0112]

[0113] Table 4 combined with KEGG pathway analysis showed that the different enriched pathways in the control group and the model group mainly included: fatty acid biosynthesis, protein digestion and absorption, lysine degradation and aminoacyl-tRNA biosynthesis (P<0.05). Table 5 shows that compared with the model group, the metabolites of tetrahydrocorticosterone, lysophosphatidic acid, 1,25-dihydroxyvitamin D3, lysophosphatidylcholine, linoleic acid, and arachidonic acid in the MLCT intervention group were downregulated, which may have an inhibitory effect on lipid synthesis to a certain extent. The metabolites of 3'-hydroxystanozolol, guanylic acid, 5-fluorouridine, 5'-adenine nucleotide, inosine, L-asparagine, L-serine, L-threonine, L-lysine, D-phenylalanine, tyrosine, and L-histidine in the MLCT group were significantly upregulated. The results show that MLCT can restore the disorder of lipid metabolism by regulating amino acid metabolism (thereby improving energy metabolism) and nucleotide metabolism. MLCT can regulate lipid metabolism by regulating the biosynthesis of phenylalanine and tyrosine. MLCT can reduce lipid metabolism disorders by regulating glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine, tyrosine metabolism, etc.

[0114] Table 6 Differential metabolites in total ion mode caused by MLCT group and olive oil group

[0115]

[0116]

[0117] As shown in Table 6, compared with the olive oil group, the metabolites of dodecanedioic acid and deoxycorticosterone in the MLCT group were significantly downregulated (P<0.05). The metabolites of 3'-hydroxystanozolol, alanyl-L-glutamine, guanosine diphosphate, guanylic acid, 5-fluorouridine, 5'-adenine nucleotide, uridine, D-glutamine, leucylproline, L-tyrosine, (5-L-glutamyl)-L-amino acid, aspartylglutamate, etc. in the MLCT group were significantly upregulated (P<0.05). The results show that MLCT can reduce the disorder of lipid metabolism by regulating purine metabolism, glutamine, tyrosine metabolism, etc.

[0118] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of MLCT in the preparation of a drug for preventing and / or treating non-alcoholic fatty liver disease, characterized in that: The MLCT is synthesized from olive oil and medium chain triglycerides.

2. Use of MLCT in the preparation of a drug for preventing and / or treating lipid metabolism disorders, characterized in that: The MLCT is synthesized from olive oil and medium chain triglycerides.

3. The use of MLCT in the preparation of a drug for improving lipid metabolism disorders in HepG2 cells, characterized in that: The MLCT is synthesized from olive oil and medium chain triglycerides.

4. The use according to any one of claims 1 to 3, characterized in that: The MLCT down-regulates the expression of FAS, ACC, SREBP-1c and PPARγ mRNA.

5. The use according to any one of claims 1 to 3, characterized in that: The MLCT up-regulated the expression of ATGL and CPT1A mRNA.

6. The use according to any one of claims 1 to 3, characterized in that: The MLCT reduces the TG and TC contents in cells.

7. The use according to any one of claims 1 to 3, characterized in that: The MLCTs regulate glycerophospholipid metabolism, purine metabolism, serine metabolism, phenylalanine and tyrosine metabolism.

8. The use according to any one of claims 1 to 3, characterized in that: In the MLCT, the content of medium-chain triglycerides is 70-85%, the distribution of long-chain fatty acids at the Sn-2 position is 80-82%, and the content of oleic acid at the Sn-2 position is 40-50%.

9. The use according to claim 8, characterized in that: The preparation method of MLCT comprises the following steps: olive oil and medium-chain triglyceride are mixed, immobilized lipase is added, a crude product is obtained by reaction, and the crude product is separated and purified to obtain MLCT; the immobilized lipase is Lipozyme TLIM.

10. A drug for preventing and / or treating lipid metabolism disorders, characterized in that: The drug uses MLCT prepared by the preparation method according to claim 9 as an active ingredient.

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