CN113564221A - Application of nicotinic acid in preparation of medicine for relieving cow's milk gland fibrosis through GPR109A receptor - Google Patents
Application of nicotinic acid in preparation of medicine for relieving cow's milk gland fibrosis through GPR109A receptor Download PDFInfo
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
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Abstract
The invention discloses an application of nicotinic acid in preparing a medicine for relieving cow's milk gland fibrosis through a GPR109A receptor, which comprises the following steps: s1, analysis of in vivo and in vitro experiments comparing the difference of GPR109A receptor expression in the mammary glands of normal cows and fibrosis-prone cows confirms that GPR109A receptor is highly expressed in the mammary glands of the mammary fibrosis cows. S2, detection of a fibrotic phenotype in cow mammary epithelial cells: specifically, the change conditions of TGF-beta 1, alpha-SMA, Collagen2, E-cadherin and the change conditions after the niacin is relieved are respectively detected in the mammary epithelial cells of the dairy cattle. The invention makes a brand-new cow's milk gland fibrosis tendency relieving strategy, separates primary cow mammary gland epithelial cells by collecting normal and mammary gland fibrosis mammary tissues, and comprehensively evaluates the relieving effect of nicotinic acid on cow's milk gland fibrosis tendency by using a molecular biological method. Nicotinic acid can effectively reduce the increase of the fibrosis phenotype in mammary epithelial cells, thereby achieving the purpose of protecting the occurrence of mammary fibrosis diseases.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to application of nicotinic acid in preparation of a medicine for relieving cow's milk gland fibrosis through a GPR109A receptor.
Background
Cow's milk gland fibrosis is a phenomenon that extracellular matrix of mammary gland tissue is accumulated in large quantity, scar tissue is proliferated, and functional parenchymal cells of mammary gland are necrotized and filled with connective tissue. The inflammatory response is a major factor in initiating the progression of the fibrotic response. Cow's milk fibrosis is a chronic progressive disease, and different types of breast diseases may develop into breast fibrosis. Mastitis is a very important disease in mammary gland diseases, and the economic loss caused by cow mastitis is up to 3000 billion yuan RMB every year in the world. Mastitis is easy to relieve, but the prevention and control of diseases after mastitis are difficult, and the prevention and control of diseases after mastitis are ignored by people in clinical production practice, so that the medicines for preventing and controlling the cow's milk fibrosis are almost zero. Anti-inflammatory drugs often do not have the function of effectively relieving fibrosis. In clinical production practice, the normal service life of one cow is about 3-5 years, and the effective milk production life is greatly shortened after the breast disease is changed, so that huge economic loss is caused. The current research on cow's milk gland fibrosis is relatively rare, and the severity is also lack of support from some basic experimental data. However, the phenomenon of cow's milk gland fibrosis is very common in clinic and is guided by cost price consideration and deficient basic research, so that the prevention and control of cow's milk gland fibrosis diseases are very important and have great difficulty. Therefore, the research and development of the anti-mammary fibrosis tendency medicine has important significance. The method for relieving the tendency of the mammary gland fibrosis to prolong the lactation period of the dairy cows is a research difficulty, and the application of the nicotinic acid as a novel medicament for resisting the tendency of the mammary gland fibrosis of the dairy cows has the characteristics of no residue and low cost, and evidence supports that the nicotinic acid can effectively relieve the tendency of the mammary gland fibrosis through GPR109A to a certain extent, so that the lactation period is prolonged.
GPR109A belongs to a G protein coupled receptor family, and is proved to be capable of inhibiting the generation and the development of inflammation by activating a downstream related signal path in previous researches, so that the anti-inflammatory effect is achieved. More importantly, GPR109A was found to have a tendency to alleviate renal fibrosis in recent literature reports. Then whether GPR109A has the function of alleviating the propensity for cow's milk fibrosis. Therefore, in the study, nicotinic acid is used as a ligand of GPR109A, the influence of GPR109A on mammary fibrosis is researched, and the nicotinic acid is applied to a cow mammary epithelial cell experiment.
Nicotinic acid, also known as niacin, belongs to a B vitamin and is a specific ligand of GPR 109A. Research shows that the traditional anti-inflammatory drugs do not have the effect of relieving fibrosis, the traditional drugs for relieving fibrosis are expensive, and other side effects such as renal toxicity and hepatotoxicity are easily generated after long-term application, so that the application of the traditional anti-inflammatory drugs and the traditional drugs for relieving fibrosis is very difficult. In recent years, research reports indicate that the nicotinic acid has the efficacy of relieving liver fibrosis to a certain extent. In addition, the nicotinic acid does not generate drug residue and is cheap and easy to obtain. Therefore, the nicotinic acid has the advantages which are not possessed by the traditional medicine as a novel medicine for resisting the tendency of the mammary gland fibrosis, but the effect of the nicotinic acid on the tendency of the mammary gland fibrosis is not clear at present, so that the nicotinic acid is supposed to be applied to relieving and preventing the tendency of the cow's mammary gland fibrosis.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides an experimental method for applying nicotinic acid to relieving cow's mammary gland fibrosis tendency through GPR109A receptor, and Western blot experiment proves that the nicotinic acid obviously inhibits TGF-beta 1, Vimentin and Collagen (II) technical scheme in the process of inhibiting mammary gland fibrosis tendency through activating GPR109A
In order to achieve the purpose, the invention provides the following technical scheme: an experimental method for applying nicotinic acid to relieve the tendency of cow's milk gland fibrosis through a GPR109A receptor comprises the following steps:
s1, collecting normal and mammary gland tissues of the milk cow with the tendency of mammary gland fibrosis, wherein the number of milk somatic cells of the fibrosis mammary gland is about 200 ten thousand/mL, and the fibrosis phenotype is increased.
S2, in vivo experiment: detection of GPR109A gene and protein levels in dairy cow mammary tissue. Total protein and total RNA were extracted from normal and fibrosis-prone milk breast tissues and used to analyze differences in GPR109A expression in normal and fibrotic tissues.
S3, in vitro experiment: detection of GPR109A gene and protein levels in bovine mammary epithelial cells. Total protein and total RNA were extracted from the untreated group of mammary epithelial cells and from mammary epithelial cells after TGF-. beta.1 stimulation and used to analyze the differential expression of GPR109A between normal and TGF-. beta.1 stimulation.
S4 test for inhibition effect of nicotinic acid on primary cow mammary epithelial cell fibrosis phenotype
Primary cow mammary epithelial cells were inoculated into 60mm × 15mm cell culture dishes, and divided into 4 groups: the method comprises the following steps of (1) performing culture for 3 hours by using a serum-free culture medium when the cell number reaches 50% of a culture dish, adding nicotinic acid, adding TGF-beta 1 after 1 hour, collecting primary cow mammary gland epithelial cells in the culture dish after 72 hours, and uniformly using the total protein content in the cells for subsequent tests by using a BCA protein concentration measuring method. In the following experiments, Western blot experiment technology is adopted to detect the protein expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in primary mammary epithelial cells.
Preferably, normal and mammary gland tissue from a cow that is predisposed to mammary fibrosis is collected, and the fibrotic mammary gland has a milk somatic cell count of about 200 ten thousand/mL and an elevated fibrotic phenotype.
Preferably, changes in GPR109A gene and protein levels in milk cow mammary tissue and mammary epithelial cells are detected in vivo and in vitro, respectively. Collection of normal and fibrosis-prone milk cow mammary tissue Total protein and Total RNA were extracted.
Preferably, the source of the mammary epithelial cells: the mammary epithelial cells of milk cow used in the in vitro experiment are derived from healthy mammary tissue of normal milk cow, and the specific method is a mammary tissue block culture method. Fresh mammary tissue blocks are obtained and washed for 3 times by sterile PBS, the fresh mammary tissue blocks are washed once by 75% alcohol, small mammary tissue blocks in the mammary tissue blocks are taken and cut into small blocks in sterile small bottles, the small mammary tissue blocks are uniformly placed in a cell culture bottle, then complete culture medium is added, and mammary epithelial cells of the dairy cow climb out around the tissue blocks in about 5 days and are used for subsequent experiments.
Preferably, the extraction method of the total protein of the mammary epithelial cell comprises the following steps: the method comprises the steps of collecting primary mammary epithelial cells by using a protein extraction reagent, extracting total protein, and detecting the expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in the mammary epithelial cells of the dairy cow by using a western blot method, thereby evaluating the effect of relieving the mammary fibrosis by the nicotinic acid.
(III) advantageous effects
Compared with the prior art, the invention provides an experimental method for relieving cow's milk gland fibrosis tendency of nicotinic acid through GPR109A receptor, which has the following beneficial effects:
1. the invention proves that GPR109A plays an important biological function in the process of fibrosis from the gene and protein level through in vivo experiments and in vitro experiments by establishing a brand-new cow's milk gland fibrosis tendency relieving strategy, and the influence of GPR109A specific agonist nicotinic acid on the fibrosis phenotype is researched by adding GPR109A specific agonist nicotinic acid in cell experiments. The relieving effect of nicotinic acid on the cow's milk gland fibrosis tendency is comprehensively evaluated by using a molecular biological method. Nicotinic acid can effectively inhibit the increase of mammary epithelial cell fibrosis phenotype induced by TGF-beta 1, thereby achieving the effects of protecting mammary gland and relieving mammary fibrosis.
2. The invention can relieve the cow's milk gland fibrosis tendency in an oral or injection mode, facilitates the relief of the cow's milk gland fibrosis tendency, only needs to put nicotinic acid into the upper layer of feed during feeding or carry out intravenous injection, is convenient and fast, has diversified administration forms, is not easy to generate drug resistance and other antibiotic pollution, does not generate stress reaction on cows, and is beneficial to the relief and prevention of the cow's milk gland fibrosis tendency.
Drawings
FIG. 1 shows GPR109A protein expression in fibrosis-prone breast tissue;
FIG. 2 shows GPR109A gene expression in fibrosis-prone breast tissue;
FIG. 3 is a TGF- β 1-induced GPR109A protein expression in bovine mammary epithelial cells;
FIG. 4 shows the TGF-. beta.1-induced GPR109A gene expression in bovine mammary epithelial cells;
FIG. 5 is a graph of the effect of nicotinic acid on TGF- β 1 protein levels in TGF- β 1-induced mammary epithelial cells;
FIG. 6 is a graph of the effect of nicotinic acid on Vimentin protein levels in TGF- β 1-induced mammary epithelial cells;
FIG. 7 is a graph of the effect of nicotinic acid on levels of Collagen2 protein in TGF- β 1-induced mammary epithelial cells;
FIG. 8 is a graph of the effect of nicotinic acid on the levels of E-cadherin protein in TGF- β 1-induced mammary epithelial cells;
FIG. 9 is a graph of the effect of nicotinic acid on α -SMA protein levels in TGF- β 1-induced mammary epithelial cells;
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.
The invention provides an experimental method for applying nicotinic acid to relieving cow's milk gland fibrosis tendency through GPR109A receptor, which comprises the following steps:
s1, collecting normal and mammary gland tissues of the milk cow with the tendency of mammary gland fibrosis, wherein the number of milk somatic cells of the fibrosis mammary gland is about 200 ten thousand/mL, and the fibrosis phenotype is increased.
S2, in vivo experiment: detection of GPR109A gene and protein levels in dairy cow mammary tissue. Total protein and total RNA were extracted from normal and fibrosis-prone milk breast tissues and used to analyze differences in GPR109A expression in normal and fibrotic tissues.
S3, in vitro experiment: detection of GPR109A gene and protein levels in bovine mammary epithelial cells. Total protein and total RNA were extracted from the untreated group of mammary epithelial cells and from mammary epithelial cells after TGF-. beta.1 stimulation and used to analyze the differential expression of GPR109A between normal and TGF-. beta.1 stimulation.
S4 test of inhibition effect of nicotinic acid on primary cow mammary epithelial cell fibrosis phenotype
Inoculating primary cow mammary epithelial cells into a cell culture dish, and dividing the primary cow mammary epithelial cells into 4 groups: the method comprises the following steps of (1) performing culture for 3 hours by using a serum-free culture medium when the cell number reaches 50% of a culture dish, adding nicotinic acid, adding TGF-beta 1 after 1 hour, collecting primary cow mammary gland epithelial cells in the culture dish after 72 hours, and uniformly using the total protein content in the cells for subsequent tests by using a BCA protein concentration measuring method. In the following experiments, Western blot experiment technology is adopted to detect the protein expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in primary mammary epithelial cells.
The screened dairy cows are characterized in that the mammary gland tissue has inflammatory reaction and history of mastitis, the number of somatic cells of the milk is about 200 ten thousand/mL, and the mammary gland tissue is hardened and hard to touch.
The detection method of the somatic cell number in the milk comprises the following steps: firstly, preparing a staining solution, and uniformly mixing 80mL of tetrachloroethane and 108mL of ethanol. The staining solution was then placed in a warm water solution at 65 ℃ and heated for 5min, and 0.6g of methyl blue was added. Then, the mixture was transferred to a refrigerator at 4 ℃ for 2 hours, and then 6mL of glacial acetic acid was added and stored at room temperature. Taking out the glass slide, dripping a 10 mu L fresh milk sample, drying in a constant temperature box, and then immersing in a staining solution to finally form a uniform glass slide sample; the prepared sample is placed in a counting plate for stained cell counting.
The mammary epithelial cells of milk cow used in the in vitro experiment are derived from healthy mammary tissue of normal milk cow, and the specific method is a mammary tissue block culture method. Fresh mammary tissue blocks are obtained and washed for 3 times by sterile PBS, the fresh mammary tissue blocks are washed once by 75% alcohol, small mammary tissue blocks in the mammary tissue blocks are taken and cut into small blocks in sterile small bottles, the small mammary tissue blocks are uniformly placed in a cell culture bottle, then complete culture medium is added, and mammary epithelial cells of the dairy cow climb out around the tissue blocks in about 5 days and are used for subsequent experiments.
The extraction method of the total protein of the mammary epithelial cells comprises the following steps: and collecting primary mammary epithelial cells by using a protein extraction reagent, extracting total protein, and detecting the protein expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in the mammary epithelial cells of the dairy cow by using a western blot method.
The observation of the effect of niacin in alleviating the propensity of cow's milk to fibrosis via the GPR109A receptor is shown below:
from figure 1 it can be seen that the protein level of GPR109A is significantly elevated in fibrotic mammary tissue compared to normal cow mammary tissue, whereas GPR109A may play a positive role in the remission of fibrotic disease.
From figure 2 it can be seen that the gene expression level of GPR109A was significantly elevated in the fibrotic mammary gland tissue compared to the normal cow mammary gland tissue, a result consistent with the intra-tissue protein results.
It can be seen from figure 3 that GPR109A protein levels were significantly elevated in cow mammary epithelial cells following TGF- β 1 stimulation, a result consistent with in vivo protein results.
From fig. 4, it can be seen that the gene level of GPR109A was significantly increased in the cow mammary epithelial cells after TGF- β 1 stimulation, and the above in vitro and in vivo results were consistent.
It can be seen from figure 5 that the specific agonist of GPR109A was effective in reducing TGF- β 1 protein levels in bovine mammary epithelial cells following nicotinic pre-treatment. TGF-beta 1 is one of the most main pathogenic factors for promoting the fibrosis process, so the nicotinic acid can effectively reduce the protein content of TGF-beta 1 in cells, thereby relieving the cow's milk gland fibrosis tendency.
It can be seen from figure 6 that the specific agonist of GPR109A was effective in reducing Vimentin levels in bovine mammary epithelial cells following nicotinic pre-treatment. Vimentin is one of the main markers of fibroblast activation, and the large-scale expression of Vimentin represents that the fibrosis process is ongoing, so that nicotinic acid can effectively reduce the protein content of Vimentin in cells, thereby relieving the cow's milk gland fibrosis tendency.
It can be seen from figure 7 that the specific agonist of GPR109A was effective in reducing the protein level of Collagen2 in cow mammary epithelial cells following nicotinic pre-treatment. Collagen2 is the major component of the extracellular matrix. The accumulation of extracellular matrix is the main reason for the formation of fibrosis, so that the nicotinic acid can effectively reduce the protein content of Collagen2 in cells, thereby relieving the tendency of cow's milk gland fibrosis.
It can be seen from figure 8 that the specific agonist of GPR109A, niacin pretreatment, was effective in increasing the protein level of E-cadherin in cow mammary epithelial cells. A decrease in the E-cadherin protein content indicates a loss of cellular polarity and an increased fibrotic process. Therefore, the nicotinic acid can effectively promote the protein content of E-cadherin in cells, thereby relieving the tendency of cow's milk gland fibrosis.
It can be seen from figure 9 that the specific agonist of GPR109A was effective in reducing the protein levels of α -SMA in cow mammary epithelial cells following nicotinic pre-treatment. alpha-SMA is one of the main markers of fibroblast activation, and the large-scale expression of the alpha-SMA represents that the fibrosis process is in progress, so that nicotinic acid can effectively reduce the protein content of the alpha-SMA in cells, thereby relieving the cow's milk gland fibrosis tendency.
In conclusion, the experiment proves that the nicotinic acid can relieve the cow's milk gland fibrosis tendency, can effectively reduce the levels of TGF-beta 1, Vimentin, Collagen2 and alpha-SMA and increase the content of E-cadherin, and thus the nicotinic acid can well relieve the cow's milk gland fibrosis tendency.
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. The application of nicotinic acid in preparing the medicine for relieving cow's milk gland fibrosis through a GPR109A receptor is characterized by comprising the following steps:
s1, collecting normal and mammary gland tissues of the dairy cow with the tendency of mammary gland fibrosis, wherein the number of milk somatic cells of the fibrosis mammary gland is about 200 ten thousand/mL, and the fibrosis phenotype is increased;
s2, in vivo experiment: detection of GPR109A gene and protein levels in dairy cow mammary tissue; collecting normal and fibrosis-prone milk cow mammary tissue total protein and total RNA were extracted for analysis of differences in GPR109A expression in normal and fibrosis tissues;
s3, in vitro experiment: detection of GPR109A gene and protein levels in dairy cow mammary epithelial cells; total protein and total RNA were extracted from the treatment-free mammary epithelial cells and from mammary epithelial cells after TGF-beta 1 stimulation, and used to analyze the differential expression of GPR109A between normal and TGF-beta 1 stimulation;
s4 test of inhibition effect of nicotinic acid on primary cow mammary epithelial cell fibrosis phenotype
Primary cow mammary epithelial cells were inoculated into 60mm × 15mm cell culture dishes and divided into 4 groups: when the cell number reaches 50% of a culture dish, a serum-free culture medium is used for replacing the culture medium to culture for 3 hours, then nicotinic acid is added, TGF-beta 1 is added after 1 hour, primary cow mammary gland epithelial cells in the culture dish are collected after 72 hours, and the total protein in the collected cells is extracted and used for the subsequent test by applying a BCA protein concentration measuring method; in the following experiments, Western blot experiment technology is adopted to detect the protein expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in primary mammary epithelial cells.
2. The use of nicotinic acid as claimed in claim 1 for preparing a medicament for alleviating cow's milk gland fibrosis through GPR109A receptor, wherein: the screened dairy cows are characterized in that the mammary gland tissue has inflammatory reaction and history of mastitis, the number of somatic cells of the milk is about 200 ten thousand/mL, and the mammary gland tissue is hardened and hard to touch.
3. The use of nicotinic acid as claimed in claim 1 for preparing a medicament for alleviating cow's milk gland fibrosis through GPR109A receptor, wherein: the detection method of the somatic cell number in the milk comprises the following steps: firstly, preparing a dyeing solution, and uniformly mixing 80mL of tetrachloroethane and 108mL of ethanol; then placing the staining solution in a warm water solution at 65 ℃ to heat for 5min, and adding 0.6g of methyl blue; then moving the mixture into a refrigerator with the temperature of 4 ℃ for 2h, adding 6mL of glacial acetic acid, and storing the mixture at normal temperature; taking out the glass slide, dripping a 10 mu L fresh milk sample, drying in a constant temperature box, and then immersing in a staining solution to finally form a uniform glass slide sample; the prepared sample is placed in a counting plate for stained cell counting.
4. The use of nicotinic acid as claimed in claim 1 for preparing a medicament for alleviating cow's milk gland fibrosis through GPR109A receptor, wherein: the mammary epithelial cells of the milk cow used in the in vitro experiment are derived from healthy mammary tissues of normal milk cows, and the specific method is a mammary tissue block culture method; fresh mammary tissue blocks are obtained and washed for 3 times by sterile PBS, the fresh mammary tissue blocks are washed once by 75% alcohol, small mammary tissue blocks in the mammary tissue blocks are taken and cut into small blocks in sterile small bottles, the small mammary tissue blocks are uniformly placed in a cell culture bottle, then complete culture medium is added, and mammary epithelial cells of the dairy cow climb out around the tissue blocks in about 5 days and are used for subsequent experiments.
5. The use of nicotinic acid as claimed in claim 1 for preparing a medicament for alleviating cow's milk gland fibrosis through GPR109A receptor, wherein: the extraction method of the total protein of the mammary epithelial cells comprises the following steps: and collecting primary mammary epithelial cells by using a protein extraction reagent, extracting total protein, and detecting the protein expression conditions of TGF-beta 1, Vimentin, Collagen2, E-cadherin and alpha-SMA in the mammary epithelial cells of the dairy cow by using a western blot method.
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