Application of propionyl carnitine in increasing content of pentadecanoic acid in mammary epithelial cells

Abstract

The invention discloses application of Propionyl carnitine (PLC) in increasing the content of pentadecanoic acid (C15: 0) in mammary epithelial cells, and belongs to the technical field of milk fat metabolism in the mammary epithelial cells, the PLC has no obvious influence on proliferation of the mammary epithelial cells of dairy cows and the total amount of triglyceride, but can cause obvious increase of the content of the C15: 0 in the BMECs, and the PLC can promote fatty acid metabolism in the BMECs, so that the content of the C15: 0 in the BMECs is increased, and the content of the C15: 0 in the BMECs is increased. According to the present invention, the C15: 0 accumulation in the milk fat is promoted by the PLC at the mouse level, such as the extension of the fatty acid chain, the fatty acid desaturation and the peroxisome fatty acid beta-oxidation, and the promotion of the C15: 0 accumulation in the milk fat by the PLC is verified at the mouse level, such that the theoretical basis is provided for the improvement of the nutritional value of the milk, and the reference basis is provided for the development of the C15: 0 product.

Description

Technical Field

[0001] The invention belongs to the technical field of milk fat metabolism in mammary epithelial cells, and specifically relates to the application of propionylcarnitine in increasing the content of pentadecanoic acid in mammary epithelial cells. Background Art

[0002] Pentadecanoic acid (C15:0), a type of odd-numbered fatty acid, has been shown to reduce the morbidity and mortality of chronic diseases in humans. It is a type of dietary fatty acid with high physiological activity and is also a potential candidate for essential fatty acids. C15:0 in nature is mainly found in milk and meat products of ruminants, and the human body synthesizes less C15:0. The content of C15:0 in milk is regulated by rumen microorganisms and the host. At present, the research on C15:0 in the mammary gland of dairy cows is mainly limited to the correlation analysis between rumen microorganisms and the content of C15:0 in milk, and the metabolic mechanism of C15:0 in the mammary gland has not yet been fully clarified. Therefore, it is of great theoretical and practical significance to explore the molecular mechanism of C15:0 synthesis in the mammary gland of dairy cows, find effective ways and methods, improve the nutritional value of milk and develop C15:0 products.

[0003] Propionylcarnitine (PLC) is a short-chain acylcarnitine that is closely related to cellular energy metabolism. Acylcarnitine can transport fatty acid acyl groups from the cytoplasm to the mitochondrial matrix for fatty acid β-oxidation, thereby generating energy to maintain cell activity. Carnitine infusion in the rumen of dairy cows can promote lipid digestion, causing an increase in the concentration of long-chain acylcarnitines in milk, a decrease in C6:0 concentration, and an increase in C16:1 concentration. Some studies have mentioned that after treating rat myocardial H9C2 cells with medium-chain acylcarnitine (myristoylcarnitine), lipid accumulation can be caused by inhibiting the AMPK / ACC / CPT1 signaling pathway, aggravating myocardial lipotoxicity. At present, research on PLC is more about studying its physiological functions through animal injection and perfusion, but there are very few studies on PLC's effects on milk fat metabolism. Summary of the invention

[0004] The present invention provides an application of propionylcarnitine in increasing the C15:0 content in bovine mammary epithelial cells (BMECs), and verifies it at the mouse level, aiming to solve the problem of how to increase the content of high-quality fatty acid C15:0 in mammary cells.

[0005] As a preferred embodiment of the present invention, the method for using propionylcarnitine to increase the C15:0 content in mammary epithelial cells is as follows: mammary epithelial cells are cultured in a complete culture medium containing 50 to 150 μM propionylcarnitine for 24 to 48 hours.

[0006] As a preferred embodiment of the use of propionylcarnitine in the present invention for increasing the C15:0 content in mammary epithelial cells, the propionylcarnitine is used to increase the viability of bovine mammary epithelial cells.

[0007] As a preferred embodiment of the use of propionylcarnitine in the present invention for increasing the C15:0 content in mammary epithelial cells, the propionylcarnitine is used to promote fatty acid metabolism in BMECs.

[0008] As a preferred embodiment of the use of propionylcarnitine of the present invention in increasing the C15:0 content in mammary epithelial cells, when the propionylcarnitine is used to promote fatty acid metabolism in BMECs, the propionylcarnitine is used to promote the elongation of fatty acid chains in fatty acid metabolism in BMECs, or / and, promote fatty acid desaturation in fatty acid metabolism in BMECs, or / and, promote peroxisomal fatty acid β-oxidation in fatty acid metabolism in BMECs.

[0009] As a preferred embodiment of the use of propionylcarnitine of the present invention in increasing the C15:0 content in mammary epithelial cells, when the propionylcarnitine is used to promote fatty acid metabolism in BMECs, the propionylcarnitine is used to promote the gene expression of long-chain fatty acid elongase 7 involved in fatty acid chain elongation in BMECs, or / and, promote the gene expression of stearoyl-CoA desaturase involved in fatty acid desaturation in BMECs, or / and, promote the gene expression of mitochondrial glycerol-3-phosphate acyltransferase in BMECs.

[0010] Beneficial effects:

[0011] 1) The present invention has found through research that propionylcarnitine can significantly increase the C15:0 content in bovine mammary epithelial cells and improve the viability of bovine mammary epithelial cells, but has no significant effect on BMECs proliferation and the total amount of triglycerides in BMECs; propionylcarnitine has no effect on the gene expression of acetyl-CoA carboxylase and fatty acid synthase involved in the de novo synthesis of fatty acids in bovine mammary epithelial cells. PLC can promote fatty acid metabolism in BMECs, such as fatty acid chain extension, fatty acid desaturation and peroxisomal fatty acid β-oxidation. Based on the present invention, it is expected to analyze the synthesis mechanism of bovine mammary C15:0, provide a theoretical basis for improving the nutritional value of milk, and also provide a reference for the development of C15:0 products.

[0012] 2) Treat with complete medium containing 100 μM propionylcarnitine dairy cow Mammary epithelial cells, after 24h of treatment, the C15:0 content in BMECs was 10-130% higher than that in the NC group.

[0013] 3) The present invention verified at the mouse level that PLC promotes the accumulation of C15:0 in milk fat. The mean value of C15:0 in milk fat in the PLC group was significantly higher than that in the NC group (P<0.01), which was 74.70% higher than that in the NC group. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a graph showing the effects of different treatment times and different PLC concentrations on BMECs activity in embodiment 1 of the present invention.

[0015] Figure 2 This is a diagram showing the effect of different concentrations of PLC on the proliferation of BMECs in Example 1 of the present invention.

[0016] Figure 3 This is a diagram showing the effect of different concentrations of PLC on BMECs apoptosis in Example 1 of the present invention.

[0017] Figure 4 The effect of 100 μM PLC on triglyceride content in BMECs in Example 2 of the present invention is shown in FIG.

[0018] Figure 5 This is a graph showing the effect of 100 μM PLC on fatty acids in BMECs in Example 3 of the present invention.

[0019] Figure 6 This is a graph showing the effect of 100 μM PLC on fatty acid metabolism genes in BMECs in Example 4 of the present invention.

[0020] Figure 7 This is a graph showing the effect of PLC on mouse milk fatty acids in Example 5 of the present invention. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is described in detail below through embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0022] The BMECs in the embodiments of the present invention are derived from primary mammary epithelial cells of Chinese Holstein cows, and specific pathogen-free SPF-grade healthy adult ICR pregnant mice are purchased from the Comparative Medicine Center of Yangzhou University. Other raw materials and detection reagents involved in the embodiments of the present invention can be purchased from the market.

[0023] Example 1

[0024] This example explores the effect of PLC on BMECs vitality, proliferation and apoptosis, and the specific steps are as follows:

[0025] 1) When BMECs were revived, complete medium (90% DMEM / F12, 10% fetal bovine serum, 100 IU / mL penicillin, 100 ug / mL streptomycin) was added to a 60 mm culture dish and the cells were cultured at 37°C, 5% CO2 and saturated humidity. When the cell density reached 80-90%, a 1:2 subculture was performed.

[0026] 2) BMECs vitality detection:

[0027] The method for detecting BMECs viability is as follows: 1×10 4 The cells were evenly spread in a 96-well plate at a density of 100 μL / well, and 100 μL of PLC complete medium of different concentrations was added to each well. The concentrations were set to 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 400 μM, and three replicates were set for each concentration. The cells were cultured in an incubator (37°C, 5% CO2). After culturing for 24 h and 48 h, CCK-8 reagent was added, and the cells were incubated at 37°C for 2 h. The absorbance was measured at a wavelength of 450 nm using an enzyme reader, and the BMECs activity was calculated according to the CCK-8 instruction manual.

[0028] After culturing BMECs with different concentrations of PLC for 24h and 48h, the results of BMECs viability detection at different treatment times are as follows: Figure 1 As shown in A, NC is the control group treated with 0 μΜ PLC. The CCK-8 test results show that different concentrations of PLC can cause a significant increase in BMECs activity (P<0.05), and the BMECs activity when the treatment time is 24h is better than that of 48h.

[0029] After BMECs were treated with different concentrations of PLC for 24 h, the CCK-8 test results were as follows: Figure 1 As shown in B, compared with NC, different concentrations of PLC had a significant effect on the activity of BMECs (P<0.05). When the PLC concentration was 50-400 μM, it promoted the cell activity. When the PLC concentration was 50 μM, the activity of BMECs was the highest.

[0030] 3) BMECs proliferation detection:

[0031] The BMECs proliferation detection method is as follows: BMECs in good condition after passaging are plated in a 96-well plate, and 2 mL of complete culture medium containing different concentrations of PLC is added to each well. The concentrations are set to 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 400 μM, and three replicates are set for each concentration; after culturing for 24 hours, BMECs are incubated with EdU reagent at 37°C for 2 hours, rinsed with PBS, fixed with paraformaldehyde at room temperature for 15 minutes, rinsed with PBS, click stained for 30 minutes, and the cell nuclei are stained with Hoechst 33342. Finally, an inverted fluorescence microscope is used to observe the cell fluorescence staining, and the proportion of positive cells is counted with Image J for statistical analysis.

[0032] The proliferation of BMECs was detected by EdU. Figure 2 As shown in A, Edu+ indicates cells that are undergoing DNA synthesis and are labeled with EdU, i.e., proliferating cells; Hoechst indicates that Hoechst is used to stain the nucleus for observing cell morphology and counting; Merge indicates that Edu+ and Hoechst staining are combined to display the cell proliferation and the distribution of the nucleus at the same time; compared with NC, there is no significant difference in the number of BMECs proliferating in different treatment groups. The cell proliferation rate was calculated by Image J as shown in Figure 2 As shown in B, it was found that different concentrations of PLC had no significant effect on the proliferation of BMECs (P>0.05).

[0033] The results of BMECs viability and BMECs proliferation assays showed that PLC at a concentration of 50-400 μM promoted cell viability, and the EdU test showed that 50-400 μM PLC did not affect BMECs proliferation. Therefore, 50-400 μM PLC was non-toxic to BMECs.

[0034] 4) BMECs apoptosis detection:

[0035] The method for detecting BMECs apoptosis is as follows: BMECs in good condition after passage are plated in a 96-well plate, and 2 mL of complete medium containing different concentrations of PLC is added to each well. The concentrations are set to 0 μM, 50 μM, 100 μM, 150 μM, 200 μM, and 400 μM, and three replicates are set for each concentration; after culturing for 24 hours, the cells are washed with PBS, and the cells are collected by trypsin digestion. After washing with PBS, BMECs are resuspended in 1×BindingBuffer, AnnexinV-FITC and propidium iodide PI are added, and incubated at room temperature in the dark for 10 minutes, 1×Binding Buffer is added, and gently mixed. Flow cytometry staining is performed within 1 hour for detection.

[0036] The results of FITCAnnexinV / PI detection of BMECs apoptosis are as follows Figure 3 As shown in A, the upper right UR represents late apoptosis, and the lower right LR represents early apoptosis. Figure 3 As shown in Figure B, compared with NC, when the concentration of PLC was ≥200 μM, the total apoptosis rate of BMECs was increased (P < 0.05); the results of early and late apoptosis were as shown in Figure 3 As shown in Figure C, compared with NC, PLC concentration had no significant effect on the early apoptosis of BMECs (P>0.05), but when the concentration of the treatment group was ≥150μM, it promoted the late apoptosis of cells (P<0.05).

[0037] Therefore, the final treatment time of 24 h and the PLC treatment concentration of 100 μM were selected for subsequent experiments.

[0038] Example 2

[0039] This example explores the effect of PLC on triglycerides in BMECs, and the specific steps are as follows:

[0040] 1) When BMECs were revived, complete medium (90% DMEM / F12, 10% fetal bovine serum, 100 IU / mL penicillin, 100 ug / mL streptomycin) was added to a 60 mm culture dish and the cells were cultured at 37°C, 5% CO2 and saturated humidity. When the cell density reached 80-90%, a 1:2 subculture was performed.

[0041] 2) The method for determining triglycerides in BMECs is:

[0042] BMECs in good condition after passage were plated in six-well plates, and 100 μL of complete culture medium containing 0 μM and 100 μM PLC were added respectively, with three replicates for each concentration. After culturing for 24 h, BMECs were digested with EDTA, washed with PBS and homogenized. After ultrasonic disruption, an appropriate amount of sample was added to a 96-well plate, and the sample was added according to the instructions of the triglyceride detection kit. The triglyceride absorbance was then measured at a wavelength of 546 nm on a microplate reader, and the intracellular protein concentration was measured using the protein concentration detection kit for calibration. The triglyceride concentration was calculated according to the formula.

[0043] The results of intracellular triglyceride content in BMECs were as follows Figure 4 As shown, 100 μM PLC had no significant effect on the triglyceride content of BMECs compared with NC (P>0.05).

[0044] Example 3

[0045] This example explores the effect of PLC on fatty acids in BMECs, and the specific steps are as follows:

[0046] 1) When BMECs were revived, complete medium (90% DMEM / F12, 10% fetal bovine serum, 100 IU / mL penicillin, 100 ug / mL streptomycin) was added to a 60 mm culture dish and the cells were cultured at 37°C, 5% CO2 and saturated humidity. When the cell density reached 80-90%, a 1:2 subculture was performed.

[0047] 2) The method for detecting fatty acids in BMECs is:

[0048] In a 60 mm culture dish containing BMECs in good condition after passage, 8 mL of complete medium containing 0 μM and 100 μM PLC was added respectively. After culturing for 24 h, the cells were washed twice with PBS, 2.5% sulfuric acid / methanol (volume / volume) solution was added, and the cells were collected in a 10 mL glass tube; the cells were crushed with a cell disruptor, in a water bath at 80°C for 1 h, 0.1 M hydrochloric acid solution and n-hexane were added, and the cells were shaken and centrifuged. The supernatant was collected in a 2 mL glass tube, anhydrous sodium sulfate was added, and the cells were allowed to stand at room temperature overnight; then the cells were shaken and centrifuged, and the supernatant was taken into a GC test bottle and determined by a mass spectrometer (GC-MS). The gas phase conditions for detecting fatty acids are: 1 μL sample, split ratio 1:10, 0.8 mL / min He gas, injector temperature 250°C, detector temperature 280°C, HP-5 chromatographic column (60 m×0.25 mm), mass spectrometry conditions: solvent delay 5 min, ionization energy 70 eV, mass number 35-500 amu.

[0049] Fatty acid test results Figure 5 As shown in the results, the C15:0 content in BMECs was 10-130% higher than that in the NC group, and its mean was significantly higher than that in the NC group (P<0.05), 48.02% higher than that in the NC group; while the C17:0 (heptadecanoic acid) and C18:0 (octadecanoic acid) contents in BMECs were significantly lower than those in the NC group (P<0.05), 58.26% and 51.21% lower than those in the NC group, respectively, and the C20:4cis-5,8,11,14 (eicosatetraenoic acid) mean was extremely significantly lower than that in the NC group (P<0.01), 60.06% lower than that in the NC group. PLC had a significant effect on the C15:0 content in BMECs, but it did not affect the accumulation of triglycerides. Triglycerides are composed of glycerol and three fatty acid molecules, so PLC can only change the fatty acid composition of triglycerides in BMECs, and does not affect the total amount of triglycerides.

[0050] Example 4

[0051] This example explores the effect of PLC on lipid metabolism genes in BMECs cells, and the specific steps are as follows:

[0052] 1) When BMECs were revived, complete medium (90% DMEM / F12, 10% fetal bovine serum, 100 IU / mL penicillin, 100 ug / mL streptomycin) was added to a 60 mm culture dish and the cells were cultured at 37°C, 5% CO2 and saturated humidity. When the cell density reached 80-90%, a 1:2 subculture was performed.

[0053] 2) Detect the expression of lipid metabolism-related genes in BMECs by fluorescence quantitative PCR. The fluorescence quantitative PCR detection method is as follows:

[0054] BMECs in good condition after passage were plated in six-well plates, and 2 mL of complete culture medium containing 0 μM and 100 μM PLC was added respectively. Three replicates were set for each concentration. After culturing for 24 hours, the culture medium was discarded and washed with PBS. The six-well plate was placed on ice, Trizol was added, and it was fully lysed. It was transferred to a 1.5 mL enzyme-free tube and chloroform was added. It was shaken vigorously, centrifuged after standing, and the supernatant was collected. An equal volume of pre-cooled isopropanol was added to mix and then stand. The supernatant was discarded after centrifugation, and 75% ethanol was added to wash the precipitate. After drying, ddH2O was added to dissolve RNA. After correcting the concentration, reverse transcription PCR was performed according to the instructions of the Takara reverse transcription kit, and then the gene expression was quantitatively detected according to the qRT-PCR kit. GAPDH was used as the internal reference, and each sample was repeated 3 times, using 2 -ΔΔCt Perform statistical analysis.

[0055] Fluorescence quantitative PCR was used to detect the expression of lipid metabolism-related genes in BMECs. Figure 6As shown in the figure, after adding 100 μM PLC, the mean gene expression level of long-chain fatty acid elongase 7 (ELOVL7) involved in fatty acid chain elongation was significantly higher than that of the NC group, 30.03% higher than that of the NC group; the mean gene expression level of stearoyl-CoA desaturase (SCD) involved in fatty acid desaturation was significantly higher than that of the NC group, 46.6% higher than that of the NC group; the mean gene expression level of mitochondrial glycerol-3-phosphate acyltransferase (GPAM) involved in triglyceride synthesis was significantly higher than that of the NC group, 70.80% higher than that of the NC group; the mean gene expression level of 17β-hydroxysteroid 17-beta dehydrogenase 4 involved in peroxisomal fatty acid β-oxidation was significantly higher than that of the NC group, 54.7% higher than that of the NC group; the mean gene expression level of 17β-hydroxysteroid 17-beta dehydrogenase 4 involved in peroxisomal fatty acid β-oxidation was significantly higher than that of the NC group, 55.3% higher than that of the NC group; the mean gene expression level of 17β-hydroxysteroid 17-beta dehydrogenase 4 involved in peroxisomal fatty acid β-oxidation was significantly higher than that of the NC group, 56.7% higher than that of the NC group; the mean gene expression level of 17β-hydroxysteroid 17-beta dehydrogenase 4 involved in peroxisomal fatty acid β-oxidation was significantly higher than that of the NC group, 54.7% higher than that of the NC group 4, HSD17B4) gene expression levels in the HSC-treated group were significantly higher than those in the NC group, 27.21% higher than those in the NC group (P<0.05); however, there was no significant effect on the gene expression levels of acetyl-coa carboxylase (ACACA) and fatty acid synthase (FASN) involved in de novo fatty acid synthesis, fatty acid binding protein 3 (FABP3) involved in fatty acid transport, and carnitine palmitoyltransferase 1A (CPT1A) involved in fatty acid β-oxidation.

[0056] Combined with the fatty acid detection results in Example 3, it can be inferred that PLC promotes mammary gland C15:0 synthesis not through the de novo fatty acid synthesis pathway, but may be caused by peroxisomal fatty acid β-oxidation.

[0057] Example 5

[0058] This example explores the effect of PLC on mouse milk fatty acids, and the specific operation is as follows:

[0059] 1) Specific pathogen-free SPF healthy adult ICR pregnant mice were randomly divided into NC group and PLC group, with 6 mice in each group. The temperature of the feeding room was 22-26℃, the humidity was 40-60%, and the lighting was 12h:12h day and night. Water and food were freely available. On the 7th day after birth, after the mice were restrained, 50μL of saline was injected into the NC group through the mouse milk duct with a microinjector, and 50μL of saline dissolved in PLC (10mg / kg·d) was injected into the PLC group through the mouse milk duct with a microinjector. The injection was once at the same time every day for 3 consecutive days. After each injection, the mice were isolated from their offspring for 2h before returning to the cage to ensure their continuous lactation function. 24h after all injections, milk samples were collected from each mouse on the same day to 2mL and frozen for fatty acid detection.

[0060] 2) Determination of fatty acids in mouse milk:

[0061] The determination method is as follows: after centrifuging the milk sample, take the upper layer of fat, put it into a glass bottle with a lid, add ether: dichloromethane: hexane (volume ratio 1:10:89), add methyl acetate, mix well, add sodium hydroxide methanol solution, vortex shake, and stand at room temperature for 10 minutes; after centrifugation, transfer the supernatant to a gas phase vial, blow dry to 0.5mL with a nitrogen blower, filter and take 1μL for gas chromatography detection.

[0062] After PLC was injected into the mammary gland of mice, the results of fatty acid detection were as follows Figure 7 As shown in the figure, the mean C15:0 in milk fat of the PLC group was significantly higher than that of the NC group (P<0.01), 74.70% higher than that of the NC group; while the mean C18:1cis9 (octadecenoic acid) and C18:3cis-9,12,15 (octadecadienoic acid) in milk fat of the PLC group were significantly lower than those of the NC group (P<0.01), 29.57% and 48.12% lower than those of the NC group, respectively. It can be seen that the proportion of C15:0 in milk fat can be significantly increased after PLC is injected into the mammary gland of mice.

[0063] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.

Claims

1. Application of propionylcarnitine in increasing the content of pentadecanoic acid in mammary epithelial cells.

2. The use of propionylcarnitine according to claim 1 in increasing the content of pentadecanoic acid in mammary epithelial cells, characterized in that: The method for increasing the content of pentadecanoic acid in mammary epithelial cells by propionylcarnitine is as follows: Mammary epithelial cells were cultured in complete culture medium containing 50-150 μM propionylcarnitine for 24 h to 48 h.

3. The use of propionylcarnitine according to claim 1 in increasing the content of pentadecanoic acid in mammary epithelial cells, characterized in that: The propionylcarnitine is used for improving the activity of cow mammary epithelial cells.

4. The use of propionylcarnitine according to claim 1 in increasing the content of pentadecanoic acid in mammary epithelial cells, characterized in that: The propionylcarnitine is used to promote fatty acid metabolism in BMECs.

5. The use of propionylcarnitine according to claim 4 in increasing the content of pentadecanoic acid in mammary epithelial cells, characterized in that: The propionylcarnitine is used to promote the elongation of fatty acid chains, or / and, promote fatty acid desaturation, or / and, promote peroxisomal fatty acid β-oxidation.

6. The use of propionylcarnitine according to claim 4 in increasing the content of pentadecanoic acid in mammary epithelial cells, characterized in that: The propionylcarnitine is used to promote the gene expression of long-chain fatty acid elongase 7 involved in fatty acid chain elongation, or / and, to promote the gene expression of stearoyl-CoA desaturase involved in fatty acid desaturation, or / and, to promote the gene expression of mitochondrial glycerol-3-phosphate acyltransferase.

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