CN113599371A - Application of metformin in preparation of medicine for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration - Google Patents
Application of metformin in preparation of medicine for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration Download PDFInfo
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Abstract
The invention relates to application of metformin in preparation of a medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration, belonging to the technical field of biological medicines. The technical scheme of the invention is that the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration is provided, the metformin has good effect of preventing thymus gland degeneration, and can be used for preparing the medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration.
Description
Technical Field
The invention relates to application of metformin in preparation of a medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration, belonging to the technical field of biological medicines.
Background
The thymus is the central immune organ that cultures T lymphocytes, and is also the organ in the body that undergoes the earliest aging-related degeneration. Thymus degeneration, which results in decreased thymocyte development and decreased numbers of primary cells, can lead to immune deficiencies, including increased incidence of infectious disease and delayed T cell reconstitution following hematopoietic stem cell transplantation, resulting in diminished protective immunity to pathogens. Prevention of thymus deterioration and promotion of thymus regeneration help to maintain and improve T cell-mediated immune homeostasis. As the population ages, extending the health life has been identified as an important goal to minimize human suffering and to enable medical systems to cope. Thus, better management of age-related degenerative regeneration of the thymus would provide benefits to the elderly population. However, the current drugs are not effective in preventing thymus degeneration or promoting regeneration of thymus tissue.
Metformin (Metformin, Met) is a biguanide drug, can improve glycemic control, and has the advantages of good safety, no hypoglycemia, reduction of cardiovascular mortality, low cost and the like. Metformin is used alone or in combination with other antidiabetic agents and has wide applications in the effective prevention of diabetes and the treatment of metabolic syndrome, obesity or polycystic ovarian disease. Metformin acts in the liver, inhibiting gluconeogenesis by blocking the mitochondrial redox shuttle. However, the mechanism of action of metformin is still poorly understood and the efficacy of the drug may be pleiotropic. For example, metformin has been found to have antioxidant and anti-aging effects, but whether metformin can improve thymus degeneration and promote tissue regeneration has not been studied.
Disclosure of Invention
The invention aims to provide a new application of metformin or pharmaceutically acceptable salt thereof, which is used for preparing a medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration.
The second purpose of the invention is to provide the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicine for preventing immune diseases caused by thymus gland degeneration.
The third purpose of the invention is to provide the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicine for changing the thymus gland structure, wherein the thymus gland structure is changed by increasing the thickness of the cortex of the thymus gland and/or making the cortex and medulla of the thymus gland have clear boundaries.
The fourth purpose of the invention is to provide application of metformin or a pharmaceutically acceptable salt thereof in preparing a medicament for reducing the number of beta-galactosidase positive cells in thymus.
The fifth purpose of the invention is to provide application of metformin or a pharmaceutically acceptable salt thereof in preparing a medicament for improving the content of CD4 positive cells and reducing the content of CD8 positive cells in thymus.
The sixth purpose of the invention is to provide application of metformin or a pharmaceutically acceptable salt thereof in preparing a medicament for promoting the expression of proteins of an autoimmune regulator and/or a sex-determining region Y box protein 2.
The seventh purpose of the invention is to provide application of metformin in preparation of medicines for promoting expression of histone acetylation lys14 and/or histone acetylation lys 27.
In order to achieve the purpose, the technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration is as follows:
application of metformin or pharmaceutically acceptable salt thereof in preparing medicaments for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration.
Compared with gallic acid, the metformin or the pharmaceutically acceptable salt thereof has better effect of preventing thymus gland degeneration, and can be used for preparing medicaments for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration.
Further, the thymus degeneration is thymus degeneration caused by thymus atrophy caused by acute senescence of D-galactose.
The technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicine for preventing the immune diseases caused by thymus gland degeneration is as follows:
application of metformin or pharmaceutically acceptable salt thereof in preparing a medicament for preventing immune diseases caused by thymus gland degeneration.
Compared with gallic acid, the metformin or the pharmaceutically acceptable salt thereof has better effect of preventing thymus gland degeneration, and can be used for preparing the medicine for preventing immune diseases caused by thymus gland atrophy.
Further, the thymus degeneration is thymus degeneration caused by thymus atrophy caused by acute senescence of D-galactose.
The technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for changing the thymus structure is as follows:
application of metformin or pharmaceutically acceptable salt thereof in preparing a medicament for changing thymus gland structure; the thymus gland structure is changed to increase the thickness of the cortex of the thymus gland and/or to make the cortex and medulla of the thymus gland have clear boundaries.
Compared with gallic acid, the metformin or the pharmaceutically acceptable salt thereof can better increase the thickness of the cortex of the thymus or enable the cortex and medulla of the thymus to be clearly demarcated, and can be used for preparing a medicine for recovering or improving the tissue structure of the thymus, in particular a medicine for increasing the thickness of the cortex of the thymus and/or enabling the cortex and medulla of the thymus to be clearly demarcated.
The technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for reducing the number of beta-galactosidase positive cells in thymus comprises the following steps:
use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the number of beta-galactosidase positive cells in the thymus.
Compared with gallic acid, the metformin or the pharmaceutically acceptable salt thereof can better reduce the number of beta-galactosidase positive cells in thymus, and can be used for preparing medicines for reducing the number of beta-galactosidase positive cells in thymus.
The technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for improving the content of CD4 positive cells in thymus and reducing the content of CD8 positive cells in thymus is as follows:
application of metformin or pharmaceutically acceptable salt thereof in preparing a medicament for increasing CD4 positive cells in thymus and reducing the content of CD8 positive cells in thymus.
The CD4 positive cell is a CD4 single positive cell (CD4+ CD8-), and the CD8 positive cell is a CD8 single positive cell (CD4-CD8 +).
Compared with gallic acid, the metformin or pharmaceutically acceptable salt thereof can better improve the content of CD4 positive cells in thymus and reduce the content of CD8 positive cells in thymus, and can be used for preparing medicines for improving the content of CD4 positive cells in thymus and reducing the content of CD8 positive cells in thymus.
The technical scheme of the application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for promoting the protein expression of the autoimmune regulatory factor and/or the sex-determining region Y box protein 2 is as follows:
use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for promoting the expression of an autoimmune regulator and/or a protein of box Y2 in the sex-determining region.
Compared with gallic acid, the metformin or the pharmaceutically acceptable salt thereof can better promote the protein expression of the Autoimmune regulator (AIRE) and/or the Sex-determining region Y-box 2(SOX2), and can be used for preparing a medicament for promoting the protein expression of the Autoimmune regulator and/or the Sex-determining region Y-box 2.
The technical scheme of the application of the metformin or pharmaceutically acceptable salt thereof in preparing the medicament for promoting the expression of histone acetylation lys14 and/or histone acetylation lys27 is as follows:
use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for promoting expression of histone acetylated lys14 and/or histone acetylated lys 27.
Compared with gallic acid, the metformin or pharmaceutically acceptable salt thereof can better promote the expression of histone acetylation lys14 and/or histone acetylation lys27, and can be used for preparing a medicine for promoting the expression of histone acetylation lys14 and/or histone acetylation lys 27.
In the use of metformin or a pharmaceutically acceptable salt thereof, preferably hydrochloride, lactate, sulfate, succinate, citrate and maleate, for the preparation of the above-mentioned medicament, the metformin pharmaceutically acceptable salt is produced by hydrolysis in the human or animal body.
Drawings
FIG. 1 is a morphological diagram of thymus obtained in Experimental example 1;
FIG. 2 is a statistical result of thymus index obtained in Experimental example 1;
FIG. 3 is a photograph of hematoxylin-eosin-stained thymus obtained in Experimental example 2;
FIG. 4 is a photograph showing staining results from detection of a change in thymic beta-galactosidase in Experimental example 3;
FIG. 5 is a statistical result of the change of thymic beta-galactosidase positive cells obtained by the repeated experiment in Experimental example 3;
FIG. 6 is a flow cytometry result of detecting changes in the thymocyte subpopulation in the thymus of control mice, wherein the abscissa is the number of CD8 cells, the ordinate is the number of CD4 cells, and the four quadrants represent different thymocyte subpopulations;
FIG. 7 is the results of flow cytometry to detect changes in the subpopulation of thymocytes in the thymus of D-gal group mice, wherein the abscissa is the number of CD8 cells, the ordinate is the number of CD4 cells, and the four quadrants represent different subpopulations of thymocytes;
FIG. 8 is the results of flow cytometry to detect changes in the subpopulations of breast cells in the thymus of the group Met r mice, wherein the abscissa is the number of CD8 cells, the ordinate is the number of CD4 cells, and the four quadrants represent different subpopulations of thymocytes;
FIG. 9 is the results of flow cytometry to detect changes in the subpopulation of thymocytes in the thymus of group Met mice, wherein the abscissa is the number of CD8 cells, the ordinate is the number of CD4 cells, and the four quadrants represent different subpopulations of thymocytes;
FIG. 10 is the flow cytometry results of detecting changes in the thymic gland cell subsets in the thymus of the GA group of mice, wherein the abscissa is the number of CD8 cells, the ordinate is the number of CD4 cells, and the four quadrants represent different thymocyte subsets;
FIG. 11 is a statistical result of the ratios of CD4 single-positive cells, CD8 single-positive cells, and CD4 single-positive cells to CD8 single-positive cells in the thymus of different mice obtained in Experimental example 4;
FIG. 12 is a graph showing the results of staining in Experimental example 5 by detecting the protein expression of mouse thymus AIRE;
FIG. 13 is a graph showing the statistical results of the protein expression levels of mouse thymus AIRE obtained by repeating the experiment in Experimental example 5;
FIG. 14 is a graph showing the results of staining in Experimental example 5 by detecting the expression of the protein of mouse thymus SOX 2;
FIG. 15 is a statistical result of the protein expression level of mouse thymus SOX2 obtained by repeating the experiment in Experimental example 5;
FIG. 16 is a band diagram of a protein strip obtained by detecting the expression of mouse thymohistone acetylation lys14 and lys27 in experimental example 6;
FIG. 17 is a statistical result of the expression level of histone acetylated lys14 obtained by repeating the experiment in Experimental example 6;
FIG. 18 is a statistical result of the expression level of histone acetylated lys27 obtained by repeating the experiment in Experimental example 6.
Detailed Description
First, experimental material
Experimental animals: 2-month-old male Kunming mice, feeding conditions: the mice were placed at 26 ℃ with a 12-hour light/dark cycle. These mice were free to obtain water and food throughout the experiment.
Experimental reagent: d-galactose (D-gal) was prepared as a solution of 120mg/kg (0.12g D-gal in 10mL of 0.9% physiological saline) using 0.9% physiological saline (0.9g of NaCl in 100mL of ultrapure water); metformin (Metformin, Met) was formulated with 0.9% physiological saline into solutions of 100mg/kg (0.1g Met in 10mL of 0.9% physiological saline) and 300mg/kg (0.3g Met in 10mL of 0.9% physiological saline); gallic Acid (GA) was used as a positive control, and a 0.5% CMC-Na solution (0.5g CMC-Na dissolved in 100mL ultrapure water) was prepared as a 250mg/kg solution (0.25g GA dissolved in 10mL 0.5% CMC-Na solution).
Second, grouping experiments
The experimental animals in the experimental examples were divided into 5 groups (10 animals each), which were: control group (control group), D-gal group, Met (first group), Met (second group), and GA group. According to the weight of the mouse, the dosage of the experimental reagent for intraperitoneal injection and gastric lavage of each g of the mouse is 0.01 mL.
Control group (control group): injecting 0.9% normal saline into abdominal cavity every day for 8 weeks;
d-gal group: d-gal solution at 120mg/kg was injected intraperitoneally daily for 8 weeks;
met group: d-gal solution at 120mg/kg was intraperitoneally injected daily for 8 weeks, starting from the third week, except for D-gal solution at 120mg/kg daily, metformin solution at 100mg/kg daily was gavaged;
met group (III): d-gal solution at a concentration of 120mg/kg is intraperitoneally injected daily for 8 weeks, and from the third week, metformin solution at a concentration of 300mg/kg is intragastrically administered daily, except for D-gal solution at a concentration of 120 mg/kg;
GA group: d-gal solution at a concentration of 120mg/kg was intraperitoneally administered daily for 8 weeks, starting from the third week, with the exception of D-gal solution at a concentration of 120mg/kg, intragastric gallic acid solution at a concentration of 250mg/kg daily.
After the experiment was completed, the mice were weighed and sacrificed, and then each group of mice was dissected, and thymus tissues of the mice were taken out, and then thymus index was measured, changes in thymus structure were observed, changes in beta-galactosidase in thymus tissues were detected, changes in thymocyte subpopulations were detected, protein expression of Autoimmune regulator (AIRE) and Sex-determining region Y-box 2(SOX determining region Y-box2, SOX2) was detected, and expression of histone acetylated lys14 and histone acetylated lys27 were detected, respectively.
Experimental example 1: the relationship between metformin and thymic index is described.
1. Experimental procedure
Thymus tissues of the removed mice (3 each per group) were photographed and thymus weights were recorded, and thymus indices were calculated as follows: the thymus index mg/g is the mouse thymus weight mg/mouse body weight g.
2. Results of the experiment
The results of the thymus morphology and thymus index obtained by the experiment are shown in fig. 1 and fig. 2, and it can be seen from fig. 2 that the thymus index of the D-gal group mice is reduced, which indicates that the thymus is atrophied, and the thymus index is increased after the metformin is administrated, wherein the thymus index of the Met r group mice is 1.3 times that of the GA group mice; the thymus index of the mouse group Met is 1.06 times that of the mouse group GA, so that the thymus index can be obviously improved by the metformin.
Therefore, the metformin has the function of preventing thymus atrophy, can be used for preparing medicaments for preventing thymus degeneration and/or promoting thymus tissue regeneration, and can also be used for preparing medicaments for preventing immune diseases caused by thymus degeneration.
Experimental example 2: relationship to metformin and the variation of thymic structure.
1. Experimental procedure
Placing a part of the thymus tissue of the taken mice (4 mice in each group) in a paraformaldehyde solution with the mass fraction of 4% (20g of paraformaldehyde is dissolved in 400mL of 0.1mol/L PBS buffer solution, adjusting the pH to 7.4, then supplementing to 500mL with ultrapure water) for fixing for 24h, then washing with running water for 6h, then performing gradient alcohol dehydration (75% alcohol overnight, 85% alcohol 1h, 95% alcohol I30 min, 95% alcohol II 30min, anhydrous alcohol I30 min, anhydrous alcohol II 30min) and xylene transparency (xylene I20 min, xylene II 20min), then performing conventional paraffin embedding, slicing treatment, dewaxing and rehydration treatment, finally staining with hematoxylin-eosin, and photographing the stained thymus tissue under a microscope (10 times).
2. Results of the experiment
The experimental hematoxylin-eosin-stained thymus tissue is shown in FIG. 3, and it can be seen from FIG. 3 that the cortex (C: cortex) and medulla (M: medula) of the thymus of the mice in the D-gal group are poorly demarcated and the cortex is significantly thinned, the thymus structures of the Met (Met) group and the GA (GA) group of the Met (Met) group are improved after the administration of metformin, the cortex and medulla demarcating (CMJ: corticosomedulary junction) of the thymus is clear, the cortex is thickened, and the structural difference of the thymus between the three groups is not significant.
Therefore, the metformin has the function of increasing the thickness of the cortex of the thymus or clearly demarcating the cortex and the medulla of the thymus, and can be used for preparing the medicine for increasing the thickness of the cortex of the thymus and/or clearly demarcating the cortex and the medulla of the thymus.
Experimental example 3: concerns the relationship between metformin and the variation in the number of beta-galactosidase positive cells.
1. Experimental procedure
Fixing a part of the thymus tissue of the mouse (4 mice in each group) in 4% paraformaldehyde solution (20g paraformaldehyde dissolved in 400mL of 0.1mol/L PBS buffer solution, adjusting pH to 7.4, and adding ultrapure water to 500mL) for 24h, washing with running water for 6h, dewatering the washed thymus tissue in 30% sucrose solution until the thymus tissue is settled at the bottom of the sucrose solution, slicing the dehydrated thymus tissue embedded with OCT on a microtome, baking the obtained frozen slice in a 60 ℃ oven for 30min, washing with PBS solution (pH 7.4) twice, adding the slice washed with PBS solution in X-gal developing solution in dark condition, incubating at 37 ℃ for 5h, and counterstaining with neutral red, finally, the film is sealed by water-soluble sealing agent and stored by taking pictures under a microscope (the magnification is 20 times and 100 times). The area occupied by beta-galactosidase positive cells was analyzed by Image J software, after which the results were statistically analyzed using GraphPad Prism 8 software. The experimental results were expressed as MEAN + -S.E.M, with P <0.05 indicating statistical significance (as compared to control, # indicates < 0.05; as compared to D-gal, # indicates P < 0.01).
2. Results of the experiment
The increase of beta-galactosidase positive cells reflects the increase of the number of aged cells of the body, and the staining pictures obtained from the experiment for detecting the change of the number of beta-galactosidase positive cells in the thymus tissue and the statistical results obtained from the 3 repeated experiments are shown in fig. 4 and 5 (the ordinate represents the percentage of the number of beta-galactosidase positive cells), and the results show that the beta-galactosidase positive cells of the mice in the D-gal group are obviously increased and mainly distributed in the cortex of the thymus. After metformin administration, the number of senescent cells (beta-galactosidase positive cells) in the thymus of mice decreased, indicating that metformin can decrease the number of beta-galactosidase positive cells, wherein the number of beta-galactosidase positive cells of mice of Met (r) group is 0.89 times that of gallic acid; the number of beta-galactosidase positive cells of the mouse group Met (II) is 0.82 times of that of the mouse group GA (gallic acid), so that the number of beta-galactosidase positive cells can be reduced more obviously by the metformin.
Therefore, the metformin has the function of reducing the number of beta-galactosidase positive cells in the thymus and can be used for preparing a medicament for reducing the number of beta-galactosidase positive cells in the thymus. However, the intrinsic mechanism by which metformin reduces the number of β -galactosidase positive cells, i.e., senescent cells (whether cells that inhibit cellular senescence or clear senescence, etc.) needs further experimental elucidation.
Experimental example 4: concerns the relationship of metformin to changes in thymocyte subpopulations.
1. Experimental procedure
The change of the thymocyte subgroup in the thymus of different groups of mice is detected by using a flow cytometer (the testing parameters are FSC410 and SSC200), and the statistical result of CD4+ CD8+, CD4+ CD8-, CD4-CD8+, CD4-CD8- (+ represents positive and-represents negative) cells in the thymus of the mice is obtained by analyzing through FlowJo software. The experimental results were expressed as MEAN ± s.e.m, with P <0.05 indicating statistical significance (as compared to the control group, <0.05, # indicating P <0.01, # indicating P < 0.001; as compared to the D-gal group, # indicating P <0.05, # indicating P <0.01, # indicating P < 0.001).
2. Results of the experiment
The change of the thymocyte subsets in the thymus of the mice of different groups is shown in fig. 6 to fig. 11, and as can be seen from fig. 6 to fig. 10 and fig. 11 (the ordinate represents the ratio of the CD4 single positive cells, the CD8 single positive cells and the CD4 single positive cells to the CD8 single positive cells), the D-gal group of mice showed a decrease in the percentage of CD4 single positive cells (CD4+ CD8-) and an increase in the percentage of CD8 single positive cells (CD4-CD8+), and the increase in the percentage of CD4 single positive cells and the decrease in the percentage of CD8 single positive cells after metformin administration, indicating that metformin can restore the disordered thymocyte subsets to normal. Among them, the amount of CD4 single-positive cells and the amount of CD8 single-positive cells decreased in the Met (r) and Met (r) group mice were comparable to those in the GA group (gallic acid) mice.
Therefore, the metformin has the functions of increasing the content of CD4 positive cells and reducing the content of CD8 positive cells in the thymus, and can be used for preparing medicaments for increasing the content of CD4 positive cells and reducing the content of CD8 positive cells in the thymus.
Experimental example 5: the relationship between metformin and protein expression of autoimmune regulator (AIRE)), sex-determining region Y box protein 2(SOX 2).
1. Experimental procedure
Placing a part of the thymus tissue of the mouse (4 mice in each group) in 4% paraformaldehyde solution (20g paraformaldehyde dissolved in 400mL of 0.1mol/L PBS buffer solution, adjusting pH to 7.4, and adding ultrapure water to 500mL) for fixation for 24h, then washing with flowing water for 6h, dehydrating with gradient alcohol (75% alcohol overnight, 85% alcohol for 1h, 95% alcohol I30 min, 95% alcohol II 30min, anhydrous alcohol I30 min, anhydrous alcohol II 30min) and xylene transparency (xylene I20 min, xylene II 20min), then embedding in paraffin, slicing, dewaxing and rehydration, placing the slices into citric acid-sodium citrate solution (6g sodium citrate and 0.8g citric acid dissolved in 2L of ultrapure water, adjusting pH to 7.4), completing antigen retrieval by a microwave mode (preheating at a medium-high fire for 5min, placing into a section for 5min at a high fire, and 10min at a medium-low fire), diluting the AIRE antibody (200 parts by mass of PBS solution with pH 7.4 is added into 1 part by mass of AIRE antibody), diluting the SOX2 antibody (700 parts by mass of PBS solution with pH 7.4 is added into 1 part by mass of SOX2 antibody), then using DAB to develop color, using hematoxylin to counter stain, then carrying out gradient alcohol dehydration (75% alcohol for 2 minutes, 85% alcohol for 2 minutes, 95% alcohol I for 2 minutes, 95% alcohol II for 2 minutes, absolute alcohol I for 2 minutes, and absolute alcohol II for 2 minutes) and xylene transparency (xylene I for 5 minutes, and xylene II for 5 minutes), finally sealing the piece with neutral gum and taking a picture under a microscope (the magnification is 20) for preservation, statistical results of protein expression levels of mouse thymus AIRE and SOX2 from 4 replicates were analyzed by Image J software. The experimental results were expressed as MEAN ± s.e.m, with P <0.05 indicating statistical significance (as compared to the control group, <0.05, # indicating P <0.01, # indicating P < 0.001; as compared to the D-gal group, # indicating P <0.05, # indicating P <0.01, # indicating P < 0.001).
2. Results of the experiment
The results of experiments for detecting the localization and expression of the autoimmune regulator (AIRE), the Y-box protein 2(SOX2) in the sex determination region of the mice in different experimental groups are shown in fig. 12 to 15, and it can be seen from fig. 12 and 13 (the ordinate is the percentage of the number of cells positive for AIRE), that the protein expression level of AIRE in the D-gal group of mice is decreased, the protein expression level of AIRE is increased after metformin administration, the protein expression level of AIRE in the Met (r) group of mice is 1.37 times that of GA (gallic acid) group of mice, and the protein expression level of AIRE in the Met (r) group of mice is 1.57 times that of GA (gallic acid) group of mice, and thus metformin can more significantly increase the expression of the protein of re AIRE, indicating that metformin can promote the protein expression of AIRE. As can be seen from fig. 14 and 15 (the ordinate is the percentage of the number of positive cells of SOX2), the protein expression level of SOX2 was decreased in the D-gal group mice compared to the normal control group, the protein expression level of SOX2 was 1.4 times that of the GA group (gallic acid) mice in the Met (r) group mice after metformin administration, and the protein expression level of SOX2 was comparable to that of the GA group (gallic acid) mice in the Met (r) group mice, and thus, metformin could more significantly increase the protein expression of SOX2, indicating that metformin could promote the protein expression of SOX 2.
Therefore, metformin has a function of promoting the expression of proteins of the autoimmune regulator and the sex-determining region Y-box protein 2, and can be used for preparing a medicament for promoting the expression of proteins of the autoimmune regulator and/or the sex-determining region Y-box protein 2.
Experimental example 6: concerning the relationship of metformin to the expression of histone acetylated lys14 and histone acetylated lys 27.
1. Experimental procedure
Freezing a part of the thymus tissue of the taken mice (3 each group) in an environment of-80 ℃, adding RIPA lysate into the frozen thymus tissue for grinding, then cracking the ground thymus tissue on ice for 30min, then centrifuging for 5min under the conditions that the temperature is 4 ℃ and the Relative Centrifugal Force (RCF) is 14000g, taking the supernatant, measuring the protein concentration by using an enzyme labeling instrument according to the specification of the BCA protein quantification kit, and adjusting the protein concentration of each group to be consistent. Firstly, boiling in water at 100 ℃ for 5min to denature proteins, and then carrying out SDS-polyacrylamide gel electrophoresis, wherein the specific process is as follows: preparing 15% separation gel, calculating protein sample loading amount, concentrating gel for 60V 30min, separating gel for 100V 40min, and wet converting to 175mA 90 min. The primary antibodies are diluted by WB primary antibody diluent according to the proportion that the lys14 antibody is diluted by 1:1000, the lys27 antibody is diluted by 1:1000, and the beta-actin antibody is diluted by 1: 2000. And finally, ECL chemiluminescence liquid is dripped for exposure, and then the statistical results of the expression levels of histone acetylation lys14 and histone acetylation lys27 of 3 repeated tests are obtained through an Image J software analysis method. The statistics were then analyzed using GraphPad Prism 8. The experimental results were expressed as MEAN ± s.e.m, with P <0.05 indicating statistical significance (as compared to the control group, # indicates <0.05, # indicates P <0.01, # indicates P < 0.001; as compared to the D-gal group, # indicates P <0.01, # indicates P < 0.001).
2. Results of the experiment
The results of experiments for detecting the expression of histone acetylated lys14 and histone acetylated lys27 in mice of different experimental groups are shown in fig. 16, fig. 17 and fig. 18, fig. 16 is a protein strip diagram of histone acetylated lys14, histone acetylated lys27 and β -actin, and from fig. 17 (the amount of protein expression of lys14 relative to internal reference) and fig. 18 (the amount of protein expression of lys27 relative to internal reference), it can be seen that the expression levels of lys14 and lys27 in mice of D-gal group are reduced, after administration of metformin, the expression level of histone acetylated lys14 in mice of Met group is 1.66 times that in mice of GA group (gallic acid), and the expression level of histone acetylated lys27 is 1.57 times that in mice of GA group (gallic acid); the expression level of histone acetylation lys14 of the mouse of Met (group) is 1.36 times that of the mouse of GA (gallic acid), and the expression level of histone acetylation lys27 is 1.23 times that of the mouse of GA (gallic acid), so that the expression of histone acetylation lys14 and lys27 can be more obviously promoted by metformin.
Therefore, metformin has a function of promoting expression of histone acetylated lys14 and histone acetylated lys27, and can be used for preparing a drug promoting expression of histone acetylated lys14 and/or histone acetylated lys 27.
Claims (7)
1. Application of metformin or pharmaceutically acceptable salt thereof in preparing medicaments for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration.
2. Application of metformin or pharmaceutically acceptable salt thereof in preparing a medicament for preventing immune diseases caused by thymus gland degeneration.
3. The application of the metformin or the pharmaceutically acceptable salt thereof in preparing the medicament for changing the thymus gland structure is characterized in that the thymus gland structure is changed by increasing the thickness of the cortex of the thymus gland and/or making the cortex and medulla of the thymus gland have clear boundaries.
4. Use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for reducing the number of beta-galactosidase positive cells in the thymus.
5. Application of metformin or pharmaceutically acceptable salt thereof in preparing a medicament for increasing CD4 positive cells in thymus and reducing the content of CD8 positive cells in thymus.
6. Use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for promoting the expression of an autoimmune regulator and/or a protein of box Y2 in the sex-determining region.
7. Use of metformin or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for promoting expression of histone acetylated lys14 and/or histone acetylated lys 27.
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| CN202111038852.6A CN113599371A (en) | 2021-09-06 | 2021-09-06 | Application of metformin in preparation of medicine for preventing thymus gland degeneration and/or promoting thymus gland tissue regeneration |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1638789A (en) * | 2001-03-15 | 2005-07-13 | 雷金纳克斯生物制药公司 | Methods of treating disorders of the eye and surrounding tissue with thymosin beta4 (t beta 4), analogues, isoforms and other derivatives |
| CN102871991A (en) * | 2012-10-24 | 2013-01-16 | 中国医学科学院放射医学研究所 | Application of dimethyldiguanide in drugs taking protective effect on chemoradiotherapy injury |
| CN103827293A (en) * | 2011-06-29 | 2014-05-28 | 通用医疗公司 | Compositions and methods for enhancing bioenergetic status in female germ cells |
-
2021
- 2021-09-06 CN CN202111038852.6A patent/CN113599371A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1638789A (en) * | 2001-03-15 | 2005-07-13 | 雷金纳克斯生物制药公司 | Methods of treating disorders of the eye and surrounding tissue with thymosin beta4 (t beta 4), analogues, isoforms and other derivatives |
| CN103827293A (en) * | 2011-06-29 | 2014-05-28 | 通用医疗公司 | Compositions and methods for enhancing bioenergetic status in female germ cells |
| CN102871991A (en) * | 2012-10-24 | 2013-01-16 | 中国医学科学院放射医学研究所 | Application of dimethyldiguanide in drugs taking protective effect on chemoradiotherapy injury |
Non-Patent Citations (4)
| Title |
|---|
| GNANAPRAGASAM ARUNACHALAM ET AL: "Metformin modulates hyperglycaemia-induced endothelial senescence and apoptosis through SIRT1", vol. 171, no. 2, pages 527 * |
| JIN LEE ET AL.: "Ursolic acid ameliorates thymic atrophy and hyperglycemia in streptozotocin-nicotinamide-induced diabetic mice", vol. 188, no. 3 * |
| 苗明三等: "玉米须总皂苷对链脲佐菌素致糖尿病大鼠肾脏、胰腺、胸腺组织细胞病变的影响", vol. 24, no. 3, pages 173 * |
| 高丹等，: "阿卡波糖联合二甲双胍对 2 型糖尿病患者炎性因子及细胞免疫功能的影响", 《实用临床医药杂志》, vol. 24, no. 21, pages 3 * |
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