CN111424008A - Experimental method for promoting animal mammary gland development by nicotinic acid - Google Patents
Experimental method for promoting animal mammary gland development by nicotinic acid Download PDFInfo
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Abstract
The invention discloses an experimental method for promoting the development of animal mammary gland by nicotinic acid, which comprises the following steps: s1, selecting 20 pregnant female mice, culturing alone, and freely eating and drinking water by adopting 12 h-12 h day-night intermittent illumination; s2, after delivery of the pregnant mice, the pregnant mice are weaned by mother mice artificially after 4 weeks of care, and the feeding conditions of two groups, namely an NT group (10) and a nicotinic acid group (10) are not changed, wherein 0.5% of nicotinic acid is added into the daily ration of the nicotinic acid group; s3, feeding the mice for 3 weeks, then carrying out cervical dislocation to kill the mice, and picking the 4 th pair of mammary glands of the mice for white mount staining. The method for promoting the development of the adolescent mammary gland by using the nicotinic acid detects the proliferation of the EPH4EV cell and the mammary gland of the mouse and the expression of the cyclin respectively through in vivo and in vitro experiments, comprehensively evaluates the effects of the nicotinic acid on the proliferation of the EPH4EV cell and the development of the mammary gland of the mouse by using molecular biology, and can remarkably promote the expression of cell proliferation markers in the cell and the mammary gland of the mouse, thereby promoting the cell proliferation and the development of the mammary gland.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to an experimental method for promoting the development of animal mammary gland by nicotinic acid.
Background
Mammary glands are the locus for the production and secretion of milk and are the only glandular organ of the offspring fed from mammals, and therefore the extent of mammary gland development directly determines the growth state of the pups. In the production process, the hypoevolutism of the mammary gland of the dairy cow can cause serious insufficiency of the lactation amount in the lactation period, direct economic loss can be caused to the livestock breeding industry, the breeding and breeding of the dairy cow can be indirectly influenced, and further the development process of the livestock breeding industry is influenced. Therefore, the study on the pubertal mammary gland development of the dairy cows and the regulation and control thereof have important significance on improving the lactation performance of animals and the growth and development of offspring thereof.
After birth, the mammary gland development stage is mainly focused on puberty, gestation, childbirth and catagen. Wherein the number of the terminal ends of the mammary ducts in adolescence directly influences the number of acinar cells in the lactation period of the animals, and has great influence on the lactation performance of the animals. Adolescent mammary gland development is mainly regulated by growth hormone, estradiol, progesterone and other hormones. In addition, nutrients also have a regulating effect on breast development, at present, the research mainly focuses on amino acids or fatty acids, but the research on vitamins necessary for organisms is rare, and partial research reports that vitamins may have a regulating effect on breast development, for example, after vitamin D3 receptor VDR is knocked out, breast ducts will overgrow. The vitamin A acid is an intermediate metabolite of the vitamin A in the animal body, and plays an important role in regulating and controlling the moderate growth of mammary glands. Meanwhile, essential vitamins are also important health-care nutrient substances for the dairy cows in perinatal period, so that the research on the vitamins is beneficial to better understanding the specific mechanism of lactation of the dairy cows and guiding the application in clinical production.
Nicotinic acid (Vitamin B3) is one of the 13 essential vitamins of the organism and is involved in the processes of lipid metabolism, tissue oxidation and respiration, glycolysis and the like in vivo. Plays an important role in the conversion process of producing acetic acid by rumen fermentation of ruminants. Adult ruminants can obtain nicotinic acid through various ways such as feed, in-vivo tryptophan conversion, rumen microbial synthesis and the like. But cannot meet the needs of intensive production and metabolism and production of high-yield cows, and the rumen acidosis must be relieved and the adaptability of animals to the external environment must be improved by adding nicotinic acid. In addition, the nicotinic acid added into the feed can obviously improve the production performance indexes of milk yield, milk fat rate and the like of the ruminant and can reduce the morbidity of ketosis, fatty liver and the like. In conclusion, the nicotinic acid has a good promoting effect on improving the economic benefit of the dairy cow breeding industry. However, no report is found about the research of the nicotinic acid on the structure, the function and the like of the mammary gland of animals, and the further popularization and application of the nicotinic acid are influenced
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an experimental method for promoting the development of animal mammary gland by nicotinic acid, which solves the problems in the background technology.
(II) technical scheme
In order to achieve the purpose, the invention provides the following technical scheme: an experimental method for promoting the development of mammary gland of animals by nicotinic acid comprises the following steps:
s1, selecting 20 pregnant female mice, culturing alone, and freely eating and drinking water by adopting 12 h-12 h day-night intermittent illumination;
s2, after the pregnant mouse parturites, the pregnant mouse is weaned by the mother mouse after caring for 4 weeks, and the feeding conditions of two groups, namely an NT group (10) and a nicotinic acid group (10) are not changed, wherein 1% of nicotinic acid is added into the daily ration of the nicotinic acid group;
s3, feeding the mice for 3 weeks, then carrying out cervical dislocation to kill the mice, and picking the 4 th pair of mammary glands of the mice for white mount staining.
Preferably, the effect of nicotinic acid on mouse mammary epithelial cells
Mouse mammary epithelial cells were cultured in 10% DMEM medium, and when the cells were overgrown in a culture flask, the cells were plated in 6 dishes using trypsinized cells, and mouse mammary epithelial cell lines were stimulated with 100uM, 500uM, 1mM, 1.5mM, and 2mM nicotinic acid (DMSO as a solvent) for two days, followed by measuring the total amount of cells using CCK8, and cell proliferation marker expression amounts (Cyclin D1 Cyclin D3 and PCNA) and cell cycle changes using Western blot and flow cytometry, respectively.
Preferably, the effect of GPR109A receptor in promoting EPH4EV cell proliferation by nicotinic acid is tested
The mouse mammary epithelial cell line EPH4EV was pretreated for 1h with 10 μ M PTX and the cells were divided into 4 groups: NT group, nicotinic acid group, PTX inhibitor group and PTX inhibitor + nicotinic acid group, then using CCK8 kit to detect the total amount of cells after incubating the cells for two days with 1mM nicotinic acid, and using Western Blot method to detect the expression level of p-AKT, p-mTOR and p-ERK1/2 protein of the cells after 1mM nicotinic acid stimulation, and observing the activation state of AKT/mTOR and ERK1/2 signal channels.
Preferably, the effect of AKT/mTOR signaling pathway in nicotinic acid-promoted mouse mammary epithelial cells is tested
The mouse mammary epithelial cell line EPH4EV was pretreated with 10 μ M MK2206(AKT inhibitor) and 200nMRapamycin (mTOR inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, an MK2206/Rapamycin inhibitor group and an inhibitor + nicotinic acid group, then using 1mM nicotinic acid for stimulation for two days, detecting the total amount of cells and the number of S-phase cells by an EDU cell proliferation detection kit and a CCK8 kit, and carrying out a Western Blot method on the cells after the stimulation by the nicotinic acid to detect the expression amount of cell proliferation markers.
Preferably, the mouse mammary epithelial cell line EPH4EV was pre-treated with 10 μ M U0126(ERK1/2 inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, a U0126 inhibitor group and an inhibitor + nicotinic acid group, wherein the total amount of cells and the number of cells in an S phase are detected by an EDU cell proliferation detection kit and a CCK8 kit after two days of stimulation by 1mM nicotinic acid, and the expression level of a cell proliferation marker is detected by a Western Blot method on the cells after stimulation by the nicotinic acid.
(III) advantageous effects
Compared with the prior art, the invention provides a method for promoting mammary gland development by nicotinic acid, which has the following beneficial effects:
1. the method for promoting the development of the adolescent mammary gland by using the nicotinic acid detects the proliferation of the EPH4EV cell and the mammary gland of the mouse and the expression of the cyclin respectively through in vivo and in vitro experiments, comprehensively evaluates the effects of the nicotinic acid on the proliferation of the EPH4EV cell and the development of the mammary gland of the mouse by using molecular biology, and can remarkably promote the expression of cell proliferation markers in the cell and the mammary gland of the mouse, thereby promoting the cell proliferation and the development of the mammary gland.
2. The niacin is orally administered, has small edible dose and no toxic or side effect, and effectively utilizes the effective components to exert the efficacy.
Drawings
FIG. 1 is a graph showing the effect of different concentrations of nicotinic acid on proliferation of mammary epithelial cells in mice according to the present invention;
FIG. 2 is a schematic diagram showing the effect of 1mM nicotinic acid on proliferation-related proteins of mammary epithelial cells of mice in the present invention;
FIG. 3 is a graph showing the activation of GPR109A on mouse mammary epithelial cells in accordance with the present invention;
FIG. 4 is a schematic representation of the effect of inhibition of the AKT signaling pathway on niacin-promoted cell proliferation in accordance with the present invention;
FIG. 5 is a schematic diagram showing the effect of AKT signaling pathway inhibition on the expression of nicotinic acid proliferation promoting markers and on downstream signaling pathways in the present invention;
FIG. 6 is a graphical representation of the effect of inhibition of the AKT signaling pathway on niacin-promoted cell numbers in the S phase;
FIG. 7 is a schematic representation of the effect of inhibition of the mTOR signaling pathway on niacin-promoted cell proliferation in accordance with the present invention;
FIG. 8 is a schematic diagram showing the effect of inhibition of the mTOR signaling pathway on the expression of a niacin proliferation-promoting marker in the present invention;
FIG. 9 is a schematic representation of the effect of inhibition of the mTOR signaling pathway on niacin-promoted S-phase cell numbers in accordance with the present invention;
FIG. 10 is a schematic representation of the effect of inhibition of the ERK signaling pathway on niacin-promoted cell proliferation in accordance with the present invention;
FIG. 11 is a schematic representation of the effect of inhibition of the ERK signaling pathway on the expression of niacin proliferation-promoting markers in accordance with the present invention;
FIG. 12 is a graphical representation of the effect of inhibition of the ERK signaling pathway on niacin-promoted number of S-phase cells in accordance with the present invention;
FIG. 13 is a graph showing the effect of 1% niacin addition to the diet on the milk ducts and the amount of TEB in adolescent mice according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-13, an experimental method for niacin-assisted development of mammary glands in animals includes the following steps:
s1, selecting 20 pregnant female mice, culturing alone, and freely eating and drinking water by adopting 12 h-12 h day-night intermittent illumination;
s2, after the pregnant mouse parturites, the pregnant mouse is weaned by the mother mouse after caring for 4 weeks, and the feeding conditions of two groups, namely an NT group (10) and a nicotinic acid group (10) are not changed, wherein 1% of nicotinic acid is added into the daily ration of the nicotinic acid group;
s3, feeding the mice for 3 weeks, then carrying out cervical dislocation to kill the mice, and picking the 4 th pair of mammary glands of the mice for white mount staining.
In order to cooperate with the above experiment, the following experimental methods are provided:
effect of nicotinic acid on mouse mammary epithelial cells
Mouse mammary epithelial cells were cultured in 10% DMEM medium, and when the cells were overgrown in a culture flask, the cells were plated in 6 dishes using trypsinized cells, and mouse mammary epithelial cell lines were stimulated with 100uM, 500uM, 1mM, 1.5mM, and 2mM nicotinic acid (DMSO as a solvent) for two days, followed by measuring the total amount of cells using CCK8, and cell proliferation marker expression amounts (Cyclin D1 Cyclin D3 and PCNA) and cell cycle changes using Western blot and flow cytometry, respectively.
Effect of GPR109A receptor in promoting EPH4EV cell proliferation by nicotinic acid
The mouse mammary epithelial cell line EPH4EV was pretreated for 1h with 10 μ M PTX and the cells were divided into 4 groups: NT group, nicotinic acid group, PTX inhibitor group and PTX inhibitor + nicotinic acid group, then using CCK8 kit to detect the total amount of cells after incubating the cells for two days with 1mM nicotinic acid, and using Western Blot method to detect the expression level of p-AKT, p-mTOR and p-ERK1/2 protein of the cells after 1mM nicotinic acid stimulation, and observing the activation state of AKT/mTOR and ERK1/2 signal channels.
Experiment of effect of AKT/mTOR signaling pathway in promoting mouse mammary epithelial cells by nicotinic acid
The mouse mammary epithelial cell line EPH4EV was pretreated with 10 μ M MK2206(AKT inhibitor) and 200nMRapamycin (mTOR inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, an MK2206/Rapamycin inhibitor group and an inhibitor + nicotinic acid group, then using 1mM nicotinic acid for stimulation for two days, detecting the total amount of cells and the number of S-phase cells by an EDU cell proliferation detection kit and a CCK8 kit, and carrying out a Western Blot method on the cells after the stimulation by the nicotinic acid to detect the expression amount of cell proliferation markers.
Experiment of effect of ERK1/2 signal channel in mouse mammary epithelial cell promoted by nicotinic acid
The mouse mammary epithelial cell line EPH4EV was pre-treated with 10 μ M U0126(ERK1/2 inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, a U0126 inhibitor group and an inhibitor + nicotinic acid group, wherein the total amount of cells and the number of cells in an S phase are detected by an EDU cell proliferation detection kit and a CCK8 kit after two days of stimulation by 1mM nicotinic acid, and the expression level of a cell proliferation marker is detected by a Western Blot method on the cells after stimulation by the nicotinic acid.
In order to cooperate with the above experimental methods, the following statistics are now provided:
as can be seen from fig. 1, nicotinic acid at a concentration of 2mM or less promoted the proliferation of EPH4EV cells, and 1mM nicotinic acid had the best proliferation of EPH4EV cells (× < 0.05, n ═ 5).
It can be seen from FIG. 2 that 1mM nicotinic acid significantly increased the expression of cell proliferation-associated markers in EPH4EV cells.
From fig. 3, it can be seen that 1mM nicotinic acid significantly activates AKT/mTOR and ERK signaling pathways (# < 0.05, n ═ 3), while 10 μ M PTX reversed nicotinic acid-AKT/mTOR and ERK signaling pathways (# < 0.05, n ═ 3).
From fig. 4, it can be seen that 10 μ M MK2206 reversed the proliferation of nicotinic acid on cells (× < 0.05, n ═ 3).
From fig. 5, it can be known that 2.5 μ M MK2206 significantly inhibits the expression of the marker for promoting cell proliferation of EPH4EV by niacin.
From FIG. 6, it can be seen that 2.5. mu.M MK2206 significantly inhibits the effect of nicotinic acid on the promotion of EPH4EV S-phase cell number.
From fig. 7, it can be seen that 200nM Rapa reversed the proliferation effect of nicotinic acid on cells (× < 0.05, n ═ 3).
From fig. 8, it can be known that 200nM Rapa significantly inhibits the expression of the marker that niacin promotes the proliferation of EPH4EV cells.
From fig. 9, it can be seen that 200nM Rapa significantly inhibited the effect of nicotinic acid on promoting the number of cells in EPH4EV S phase.
From fig. 10, it can be seen that 10 μ M U0126 reversed the proliferation of nicotinic acid on cells (× < 0.05, n ═ 3).
From fig. 11, it can be seen that 10 μ M U0126 significantly inhibits the expression of the marker for promoting cell proliferation of EPH4EV by nicotinic acid.
From fig. 12, it can be seen that 10 μ M U0126 significantly inhibits the effect of nicotinic acid on promoting the number of cells in EPH4EV S phase.
It can be seen from fig. 13 that 0.5% of nicotinic acid contained in drinking water significantly promoted the number of ductus mammae and ductus lactiae terminal ends.
Taken together, nicotinic acid promotes mammary gland development in mice by activating the AKT/mTOR and ERK signaling pathways.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a qualifier" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. An experimental method for promoting the development of mammary glands of animals by nicotinic acid, which is characterized by comprising the following steps:
s1, selecting 20 pregnant female mice, culturing alone, and freely eating and drinking water by adopting 12 h-12 h day-night intermittent illumination;
s2, after delivery of the pregnant mice, the pregnant mice are weaned by mother mice artificially after 4 weeks of care, and the feeding conditions of two groups, namely an NT group (10) and a nicotinic acid group (10) are not changed, wherein 0.5% of nicotinic acid is added into the daily ration of the nicotinic acid group;
s3, feeding the mice for 3 weeks, then carrying out cervical dislocation to kill the mice, and picking the 4 th pair of mammary glands of the mice for white mount staining.
2. The experimental method for the niacin enhancement of the mammary gland development of animals according to claim 1, wherein:
effect of nicotinic acid on mouse mammary epithelial cells
Mouse mammary epithelial cells were cultured in 10% DMEM medium, and when the cells were overgrown in a culture flask, the cells were plated in 6 dishes using trypsinized cells, and mouse mammary epithelial cell lines were stimulated with 100uM, 500uM, 1mM, 1.5mM, and 2mM nicotinic acid (DMSO as a solvent) for two days, followed by measuring the total amount of cells using CCK8, cell proliferation marker expression amounts (Cyclin D1, Cyclin D3, and PCNA) and cell cycle changes using Western blot and flow cytometry, respectively.
3. The experimental method for the niacin enhancement of the mammary gland development of animals according to claim 1, wherein:
effect of GPR109A receptor in promoting EPH4EV cell proliferation by nicotinic acid
The mouse mammary epithelial cell line EPH4EV was pretreated for 1h with 10 μ M PTX and the cells were divided into 4 groups: NT group, nicotinic acid group, PTX inhibitor group and PTX inhibitor + nicotinic acid group, then using CCK8 kit to detect the total amount of cells after incubating the cells for two days with 1mM nicotinic acid, and using Western Blot method to detect the expression level of p-AKT, p-mTOR and p-ERK1/2 protein of the cells after 1mM nicotinic acid stimulation, and observing the activation state of AKT/mTOR and ERK1/2 signal channels.
4. The experimental method for the niacin enhancement of the mammary gland development of animals according to claim 1, wherein:
experiment of effect of AKT/mTOR signaling pathway in promoting mouse mammary epithelial cells by nicotinic acid
The mouse mammary epithelial cell line, EPH4EV, was pretreated with 10. mu.M MK2206(AKT inhibitor) and 200nM Rapamycin (mTOR inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, an MK2206/Rapamycin inhibitor group and an inhibitor + nicotinic acid group, then using 1mM nicotinic acid for stimulation for two days, detecting the total amount of cells and the number of S-phase cells by an EDU cell proliferation detection kit and a CCK8 kit, and carrying out a Western Blot method on the cells after the stimulation by the nicotinic acid to detect the expression amount of cell proliferation markers.
5. The experimental method for the niacin enhancement of the mammary gland development of animals according to claim 1, wherein:
experiment of effect of ERK1/2 signal channel in mouse mammary epithelial cell promoted by nicotinic acid
The mouse mammary epithelial cell line EPH4EV was pre-treated with 10 μ M U0126(ERK1/2 inhibitor) for 1h and the cells were divided into 4 groups: and (3) an NT group, a nicotinic acid group, a U0126 inhibitor group and an inhibitor + nicotinic acid group, wherein the total amount of cells and the number of cells in an S phase are detected by an EDU cell proliferation detection kit and a CCK8 kit after two days of stimulation by 1mM nicotinic acid, and the expression level of a cell proliferation marker is detected by a Western Blot method on the cells after stimulation by the nicotinic acid.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111772049A (en) * | 2020-08-19 | 2020-10-16 | 四川农业大学 | Additive compound package for promoting growth of replacement gilt mammary gland and application thereof |
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| CN110376366A (en) * | 2019-07-19 | 2019-10-25 | 吉林大学 | A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor |
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| CN101962645A (en) * | 2010-08-26 | 2011-02-02 | 东北农业大学 | Method for promoting growth and lactation of mammary epithelial cells of milk cow |
| CN110376366A (en) * | 2019-07-19 | 2019-10-25 | 吉林大学 | A kind of niacin is applied to the experimental method for the treatment of mastadenitis of cow by GPR109A receptor |
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| CN111772049A (en) * | 2020-08-19 | 2020-10-16 | 四川农业大学 | Additive compound package for promoting growth of replacement gilt mammary gland and application thereof |
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