CN113637731B - Method for regulating milk protein and milk fat synthesized by bovine mammary gland by utilizing beta-sitosterol - Google Patents

Method for regulating milk protein and milk fat synthesized by bovine mammary gland by utilizing beta-sitosterol Download PDF

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CN113637731B
CN113637731B CN202110813551.XA CN202110813551A CN113637731B CN 113637731 B CN113637731 B CN 113637731B CN 202110813551 A CN202110813551 A CN 202110813551A CN 113637731 B CN113637731 B CN 113637731B
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sitosterol
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金永成
刘馨璐
沈景林
宗金鑫
刘浃怡
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Jilin University
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Abstract

The invention relates to the technical field of synthesis of milk proteins and milk fat in cow mammary epithelial cells, in particular to a method for regulating synthesis of milk proteins and milk fat from cow mammary by using beta-sitosterol, which comprises the following steps: step 1: inoculating MAC-T cells into a culture dish containing a growth medium for culture; step 2: performing differentiation induction on the MAC-T cells, simultaneously adding beta-sitosterol with different concentrations, and collecting RNA and protein; step 3: detecting the regulation and control effect of beta-sitosterol on mammary epithelial cells by qPCR and Western Blot; step 4: the regulation mechanism of beta-sitosterol on the lactation function of mammary epithelial cells is discussed by qPCR and Western Blot methods, and the method for regulating milk protein and milk fat synthesis of cow mammary gland by using the beta-sitosterol can improve the production capacity of milk protein and milk fat synthesis of mammary epithelial cells and improve the milk fat composition.

Description

Method for regulating milk protein and milk fat synthesized by bovine mammary gland by utilizing beta-sitosterol
Technical Field
The invention relates to the technical field of synthesis of milk proteins and milk fat in cow mammary gland epithelial cells, in particular to a method for regulating synthesis of milk proteins and milk fat by utilizing beta-sitosterol.
Background
Cows are one of important economic animals in livestock breeding industry in China, and although the milk industry in China is gradually growing, all aspects of the cows reach international standards, the cows still have a lifting space compared with the countries with developed milk industry. The multiple pharmacological actions of beta-sitosterol also lead to the wide research of the beta-sitosterol as a novel green additive, and the agricultural department of China listed the phytosterol as a novel functional feed additive in 2008, which allows the phytosterol to be added in the cultivation of poultry and growing-finishing pigs, but the application of the phytosterol as a feed additive in the dairy cow cultivation is not allowed. Beta-sitosterol, which is one of the phytosterols, has been allowed to be added in the cultivation of growing-finishing pigs and poultry, however, the application of beta-sitosterol of a single component in the dairy cow cultivation is rarely reported.
In the existing dairy cow breeding process, the influence effect of beta-sitosterol with different concentrations on milk synthesis by adding beta-sitosterol into dairy cow mammary epithelial cells cannot be judged, so that the effect is unfavorable for related staff to use the beta-sitosterol as a feed additive according to needs, and the yield and quality of milk are reduced, and therefore, aiming at the current situation, a method for adjusting the milk protein and the milk fat synthesized by the dairy cow mammary by using the beta-sitosterol is urgently needed to be developed so as to overcome the defects in the current practical application.
Disclosure of Invention
The invention aims to provide an application effect of beta-sitosterol in promoting lactation of cows, and discloses a mechanism for enhancing the lactation function of mammary epithelial cells of the cows, so that the beta-sitosterol can be used as a novel feed additive in lactation production of cows or dairy goats.
In order to achieve the above purpose, the present invention provides the following technical solutions:
a method for regulating milk protein and milk fat synthesized by bovine mammary gland by using beta-sitosterol, which comprises the following steps:
step 1: inoculating MAC-T cells into a culture dish containing a growth medium, and culturing in a cell culture box at 37 ℃;
step 2: performing differentiation induction on the MAC-T cells for three days, adding beta-sitosterol with different concentrations, and extracting and collecting RNA and protein after three days;
step 3: detecting the regulation and control effects of beta-sitosterol on mammary epithelial cell beta-casein, signal pathway PI3K/Akt/mTOR, JAK2-STAT5 and milk fat synthesis related enzyme by qPCR and Western Blot method;
step 4: the regulation and control mechanism of beta-sitosterol on the lactation function of mammary epithelial cells is primarily discussed through qPCR and Western Blot methods.
In summary, the invention has the following beneficial effects:
by quantitatively detecting the expression level of milk fat milk protein synthesis related genes by utilizing real-time fluorescence and detecting related proteins by utilizing Western blotting, the method discovers that beta-sitosterol with the concentration of 0.1-10 mu M promotes the synthesis of beta-casein in milk cow mammary epithelial cells, enhances the lactation function of the milk cow mammary epithelial cells, and beta-sitosterol activates PI3K/Akt/mTOR, JAK2-STAT5 and promotes the expression of related cytokines by regulating GH-IGF1 axis and HIF-1α, improves the lactation function of milk cow mammary gland, increases economic benefit, thus confirming that the beta-sitosterol with the low concentration can promote the synthesis of beta-casein in the milk cow mammary epithelial cells and improve the expression of milk fat synthesis genes and protein, has the effect of enhancing the lactation function of the milk cow mammary epithelial cells, and on the other hand, the beta-sitosterol with the high concentration (more than 30 mu M) can inhibit the synthesis of milk cow mammary epithelial cells, and promote the expression of fat synthesis genes, thereby revealing that the beta-sitosterol can be used as a feed additive or a feed ingredient to be applied to the lactation of milk cow mammary epithelial cells in the final stage, can be promoted, and the milk synthesis of milk cow mammary epithelial cells can be promoted, and the milk quality of cow milk quality can be promoted at the end stage is promoted, and the milk yield is promoted to be promoted fast and promoted to promote the milk quality of milk quality is promoted to be promoted relatively promoted to be popularized to relevant milk quality.
Drawings
FIG. 1 is a graph showing the effect of varying concentrations of beta-sitosterol on bovine mammary epithelial cell beta-casein expression in an example of the invention.
FIG. 2 is a graph showing the effect of different concentrations of beta-sitosterol on the intracellular PI3K/AkT1/mTOR, JAK2-STAT5 signaling pathway in bovine mammary epithelial cells in an example of the invention.
FIG. 3 is a graph showing the effect of different concentrations of beta-sitosterol on milk fat synthesis related factors of bovine mammary epithelial cells in an embodiment of the present invention.
FIG. 4 is a graph showing the effect of varying concentrations of beta-sitosterol on bovine mammary epithelial GH-IGF1 axis expression in accordance with an embodiment of the invention.
FIG. 5 is a graph showing the effect of varying concentrations of beta-sitosterol on bovine mammary epithelial cell HIF-1 alpha and downstream factor EPO expression in an embodiment of the invention.
FIG. 6 is a graph showing the effect of varying concentrations of beta-sitosterol on the expression of the socs family in bovine mammary epithelial cells in an example of the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Specific implementations of the invention are described in detail below in connection with specific embodiments.
Referring to fig. 1-6, the method for adjusting milk protein and milk fat synthesized by bovine mammary gland by using beta-sitosterol provided by the embodiment of the invention comprises the following steps:
step 1: inoculating MAC-T cells into a culture dish containing a growth medium, and culturing in a cell culture box at 37 ℃;
step 2: performing differentiation induction on the MAC-T cells for three days, adding beta-sitosterol with different concentrations, and extracting and collecting RNA and protein after three days;
step 3: detecting the regulation and control effects of beta-sitosterol on mammary epithelial cell beta-casein, signal pathway PI3K/Akt/mTOR, JAK2-STAT5 and milk fat synthesis related enzyme by qPCR and Western Blot method;
step 4: the regulation and control mechanism of beta-sitosterol on the lactation function of mammary epithelial cells is primarily discussed through qPCR and Western Blot methods.
By quantitatively detecting the expression level of milk fat milk protein synthesis related genes by utilizing real-time fluorescence and detecting related proteins by utilizing Western blotting, the method discovers that beta-sitosterol with the concentration of 0.1-1 mu M promotes the synthesis of beta-casein in milk cow mammary epithelial cells, enhances the lactation function of the milk cow mammary epithelial cells, beta-sitosterol with the low concentration can promote the synthesis of beta-casein in milk cow mammary epithelial cells and improve milk fat synthesis genes and protein expression, beta-sitosterol with the high concentration (more than 30 mu M) can inhibit the milk synthesis of milk cow mammary epithelial cells, so that beta-sitosterol can be used as a feed additive or a component contained in feed raw materials to be applied to the feeding process of milk cows, can inhibit the milk synthesis in the lactation period and promote quick milk drying, is beneficial to improving the yield and quality of milk, and provides convenience for related workers.
In one embodiment of the present invention, in step 1, the culturing of the mammary epithelial cells of the dairy cows comprises the following specific steps:
MAC-T cells were cultured in a growth medium of DMEM,10% FBS, 5. Mu.g/ml insulin, 1. Mu.g/ml hydrocortisone, 1% penicillin-streptomycin, and after 2-3 passages, the MAC-T cells were inoculated in six well plates containing 2ml differentiation medium for differentiation induction for 3 days, the differentiation medium was DMEM,10% FBS, 5. Mu.g/ml insulin, 1. Mu.g/ml hydrocortisone, 5. Mu.g/ml prolactin, 1% penicillin-streptomycin, and different concentrations of beta-sitosterol were added to culture, the concentrations of beta-sitosterol were 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, respectively.
In the process of culturing bovine mammary epithelial cells (MAC-T), inoculating the mammary epithelial cells in a growing period to a six-hole plate, adopting a growth culture solution for standing culture of each hole until the cells grow to about 70%, and then entering the next process, wherein 1% penicillin-streptomycin is prepared by 100IU/ml penicillin and 100 mug/ml streptomycin.
In one embodiment of the present invention, referring to fig. 1, in step 2, the six-well plate containing the differentiation medium is subjected to the following grouping process:
treatment group: absorbing and discarding the culture solution, cleaning with PBS, adding differentiation culture solution containing beta-sitosterol with different concentrations, and culturing in a cell incubator at 37 ℃ for 3 days;
control group: sucking and discarding the culture solution, cleaning with PBS, adding the differentiation culture solution, and culturing in a cell incubator at 37 ℃ for 3 days;
after each group is cultured for 3 days, cow mammary gland epithelial cells are collected, RNA and protein are extracted, and qPCR and western blot are utilized to detect the expression of beta-casein.
The culture solutions used in the treatment group and the control group were changed every day, and the results showed that the treatment group added with β -sitosterol (0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M) significantly promoted the expression of β -casein mRNA (P < 0.05) and the small concentration of β -sitosterol (0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M) significantly improved the expression of β -casein mRNA in the treatment group (P < 0.01) compared to the control group by detecting the expression of β -casein using qPCR, western blot as shown in A of FIG. 1. We selected the drug groups of 0.1. Mu.M, 1. Mu.M, 10. Mu.M, 30. Mu.M beta-sitosterol and the control group for comparison of beta-casein expression, and the results are shown in FIG. 1B, which are consistent with the above-mentioned gene expression. The results show that 0.1. Mu.M, 1. Mu.M, 10. Mu.M beta-sitosterol can increase the expression of bovine mammary epithelial cell (MAC-T) beta-casein.
In one embodiment of the present invention, referring to fig. 2, in step 3, the effect of β -sitosterol on the milk cow mammary epithelial cell milk protein synthesis-related pathway includes:
the qPCR and western blot detection of the JAK2-STAT5 of the MAC-T cell lactation signal path and the qPCR and western blot detection of the PI3K/AKT1/mTOR of the MAC-T cell lactation signal path are respectively carried out by inoculating the cells into six pore plates, dividing the six pore plates into 5 groups, namely a control group and a beta-sitosterol medicine group, and respectively selecting the concentrations of the 3d and beta-sitosterol medicine groups from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
qPCR detects the effect of JAK2, ELF5 and STAT5 gene expression in 5 different groups, and the result shows that 1 mu M beta-sitosterol can greatly improve AK2, ELF5 and STAT5 gene expression (P < 0.01) as shown in A of figure 2; the western blot detects the effect of phosphorylated STAT5 protein expression in 5 different groups, and the result is shown in C of fig. 2, where the protein level is consistent with the gene expression; qPCR (quantitative polymerase chain reaction) detects the influence of PI3K/AKT1/mTOR (mTOR) pathway related gene expression in 5 different groups, and the result shows that 1 mu M beta-sitosterol can obviously improve mTOR gene expression (P < 0.05) as shown in B of figure 2; the S6K1 gene expression is extremely obviously improved (P < 0.01). The western blot detects the effect of mTOR, P-mTOR, S6K1 and P-S6K1 protein expression in 5 different groups, and the result is that as shown in D of figure 2, 1 mu M beta-sitosterol significantly improves mTOR, P-mTOR and P-S6K1 protein expression (P < 0.05); these findings indicate that β -sitosterol affects phosphorylated STAT5- β, P-mTOR, P-S6K1 levels by activating JAK2-STAT5, mTOR signaling pathway, and ultimately regulates β -casein expression.
In one embodiment of the present invention, referring to fig. 3, in step 4, the effect of β -sitosterol on milk fat synthesis related cytokines of dairy cow mammary epithelial cells includes:
the method for qPCR detection and western blot detection of the cell line-associated cytokine of the milk fat synthesis of the MAC-T cell is to inoculate the cell line into a six-hole plate, divide the cell line into 5 groups, respectively a control group and a beta-sitosterol medicine group, and treat the cell line-associated cytokine for 3d, wherein the concentrations of the beta-sitosterol medicine group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
qPCR detects the effect of fatty acid uptake gene (LPL), activation and transport gene (FASN, ACACA, SCD) expression in 5 different groups; the effect of the beta-sitosterol on the expression of fatty acid network regulatory genes (SREBP 1, PPARalpha and the like) is detected, and the result is that as shown in A of figure 3, 10 mu M beta-sitosterol obviously improves the expression level of ACC and PSMA5 genes (P < 0.05); very significantly improving the expression level of SCD, SREBP1 and LPL genes (P < 0.01); the western blot detects the protein level effects of SCD, PSMA5, SREBP1 and PPARα in 5 different groups, and the result is that as shown in B of figure 3, 10 mu M beta-sitosterol significantly increases the protein level of PSMA5 (P < 0.05); very significantly increasing SCD, pparα protein levels (P < 0.01); these findings indicate that β -sitosterol affects milk fat synthesis related enzymes as well as network regulatory factors, ultimately regulating mammary epithelial lactation capacity.
In one embodiment of the present invention, referring to fig. 4, in step 4, the effect of β -sitosterol on milk protein-related gene expression comprises:
the detection method comprises inoculating cells into six-hole plates, dividing into 5 groups, respectively a control group and a beta-sitosterol drug group, and treating for 3 days, wherein the concentrations of the beta-sitosterol drug group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
The effect of GH-IGF1 axis-associated gene expression in 5 different groups was examined by qPCR, with the result that 1 μΜβ -sitosterol significantly increased the gene expression of GH, GHR, IGF1 (P < 0.01) as shown in fig. 4; significantly reduced IGFBP1 gene expression (P < 0.05).
In one embodiment of the present invention, referring to fig. 5, in step 4, the effect of β -sitosterol on expression of milk fat synthesis-related factors comprises:
the detection method comprises inoculating cells into six-well plates, dividing into 5 groups, namely a control group and a beta-sitosterol drug group, and treating for 3 days, wherein the concentration of the beta-sitosterol drug group is respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
The effect of HIF-1 alpha related gene expression in 5 different groups is detected by qPCR, and the result is that as shown in A of figure 5, 10 mu M beta-sitosterol extremely and obviously improves the gene expression of HIF-1 alpha and EPO (P < 0.01); gene expression of EPOR was significantly improved (P < 0.05). Then the protein level expression change of HIF-1 alpha is detected by western blot, and the result shows that the protein expression of HIF-1 alpha is remarkably improved by 10 mu M beta-sitosterol (P < 0.01) as shown in the B of figure 5.
In one embodiment of the invention, referring to fig. 6, in step 4, the effect of β -sitosterol on milk fat milk protein synthesis includes:
the detection method comprises inoculating cells into six-hole plate, dividing into 5 groups, respectively, wherein the control group and the beta-sitosterol drug group are respectively used for treating for 3 days, and the concentrations of the beta-sitosterol drug group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
The effect of SOCS2 and SOCS3 related gene expression in 5 different groups was detected by qPCR, and as a result, 1 mu M beta-sitosterol significantly reduced SOCS2 and SOCS3 gene expression (P < 0.05) as shown in A of FIG. 6; then, the protein level expression changes of SOCS2 and SOCS3 are detected by western blot, and the result shows that as shown in B of figure 6, 1 mu M beta-sitosterol extremely and obviously reduces SOCS2 protein expression (P < 0.01), and 10 mu M beta-sitosterol obviously inhibits SOCS3 protein expression (P < 0.05).
In conclusion, according to the invention, beta-sitosterol with different concentrations is added to dairy cow mammary epithelial cells (MAC-T), in-vitro cell culture is carried out in a culture medium, the regulation mechanism of the beta-sitosterol on the lactation function of the dairy cow mammary epithelial cells can be obtained initially through qPCR and detection of a Western Blot method, the production capacity of the dairy cow mammary epithelial cells can be obviously improved by adding the beta-sitosterol with the range of 0.1-10 mu M, the milk fat synthesis is improved, the milk fat composition is improved, the synthesis of the dairy cow milk proteins and milk fat in the dairy cow mammary epithelial cells can be inhibited by the beta-sitosterol with high concentration (more than 30 mu M), the synthesis effect of milk is inhibited, the rapid milk drying effect is achieved, the expression of SOCS2 and SOCS3 can be inhibited, the synthesis of the milk proteins can be promoted, the expression of HIF1α can be promoted, and the milk fat synthesis can be improved by activating the HIF channel.
It should be noted that the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, it is possible to make several modifications and improvements without departing from the concept of the present invention, and these should also be regarded as the scope of protection of the present invention, and these should not affect the effect of the implementation of the present invention and the practical applicability of the patent.

Claims (5)

1. A method for regulating milk proteins and milk fat synthesized by bovine mammary gland by using beta-sitosterol, which is characterized by comprising the following steps:
step 1: inoculating MAC-T cells into a culture dish containing a growth medium, and culturing in a cell culture box at 37 ℃;
step 2: performing differentiation induction on the MAC-T cells for three days, adding beta-sitosterol with different concentrations, and extracting and collecting RNA and protein after three days;
step 3: detecting the influence of beta-sitosterol on the protein expression and gene expression level of mammary epithelial cells beta-casein, signal paths PI3K/Akt/mTOR and signal paths JAK2-STAT5 by qPCR and Western Blot methods;
step 4: the influence of the beta-sitosterol on the lactation function of the mammary epithelial cells is detected by qPCR and Western Blot methods, wherein the influence of the beta-sitosterol on the lactation function of the mammary epithelial cells comprises the following steps: effect of beta-sitosterol on milk protein related gene expression, expression of beta-sitosterol on milk fat synthesis related factor, and beta-sitosterol on milk fat milk protein synthesis;
among them, the effects of β -sitosterol on milk protein-related gene expression include:
the detection method comprises inoculating cells into six-hole plates, dividing into 5 groups, namely a control group and a beta-sitosterol drug group, and treating for 3 days, wherein the concentrations of the beta-sitosterol drug group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M;
wherein the effect of beta-sitosterol on expression of milk fat synthesis-related factors comprises:
the detection method comprises inoculating cells into six-well plates, dividing into 5 groups, namely a control group and a beta-sitosterol drug group, and treating 3d, wherein the concentration of the beta-sitosterol drug group is respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M;
among these, the effects of β -sitosterol on milk fat milk protein synthesis include:
the detection method comprises inoculating cells into six-hole plate, dividing into 5 groups, respectively, wherein the control group and the beta-sitosterol drug group are respectively used for treating for 3 days, and the concentrations of the beta-sitosterol drug group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
2. The method for regulating synthesis of milk proteins and milk fat by using beta-sitosterol according to claim 1, wherein in step 1, the specific steps of culturing the mammary epithelial cells of the cows are:
MAC-T cells were cultured in a growth medium of DMEM,10% FBS, 5. Mu.g/ml insulin, 1. Mu.g/ml hydrocortisone, 1% penicillin-streptomycin, and after 2-3 passages, the MAC-T cells were inoculated in six well plates containing 2ml differentiation medium for differentiation induction for 3 days, the differentiation medium was DMEM,10% FBS, 5. Mu.g/ml insulin, 1. Mu.g/ml hydrocortisone, 5. Mu.g/ml prolactin, 1% penicillin-streptomycin, and different concentrations of beta-sitosterol were added to culture, the concentrations of beta-sitosterol were 0.01. Mu.M, 0.1. Mu.M, 1. Mu.M, 5. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, respectively.
3. The method for regulating milk proteins and fats from bovine mammary gland according to claim 2, wherein in step 2, the six-well plate containing the differentiation medium is subjected to the following grouping treatment:
treatment group: absorbing and discarding the culture solution, cleaning with PBS, adding differentiation culture solution containing beta-sitosterol with different concentrations, and culturing in a cell incubator at 37 ℃ for 3 days;
control group: sucking and discarding the culture solution, cleaning with PBS, adding the differentiation culture solution, and culturing in a cell incubator at 37 ℃ for 3 days;
after each group is cultured for 3 days, cow mammary gland epithelial cells are collected, RNA and protein are extracted, and qPCR and western blot are utilized to detect the expression of beta-casein.
4. A method for regulating milk protein and milk fat synthesis in the mammary gland of cows by β -sitosterol as claimed in claim 3 wherein in step 3 the effect of β -sitosterol on the milk protein synthesis-related pathways in the mammary epithelial cells of cows comprises:
the qPCR and western blot detection of the JAK2-STAT5 of the MAC-T cell lactation signal path and the qPCR and western blot detection of the PI3K/AKT1/mTOR of the MAC-T cell lactation signal path are respectively carried out by inoculating the cells into six pore plates, dividing the six pore plates into 5 groups, namely a control group and a beta-sitosterol medicine group, and respectively selecting the concentrations of the 3d and beta-sitosterol medicine groups from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
5. The method of using β -sitosterol for regulating milk protein and milk fat synthesis in the mammary gland of cows according to any of claims 1 to 4, wherein the effect of β -sitosterol on the milk fat synthesis associated cytokines in the mammary epithelial cells of cows in step 4 comprises:
the method for qPCR detection and western blot detection of the cell line-associated cytokine of the milk fat synthesis of the MAC-T cell is to inoculate the cell line into a six-hole plate, divide the cell line into 5 groups, respectively a control group and a beta-sitosterol medicine group, and treat the cell line-associated cytokine for 3d, wherein the concentrations of the beta-sitosterol medicine group are respectively selected from 0.1 mu M,1 mu M,10 mu M and 30 mu M.
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