CN118021781A - Application of small molecular compound in preparing medicament for treating or improving pituitary prolactin cell aging and promoting prolactin secretion - Google Patents
Application of small molecular compound in preparing medicament for treating or improving pituitary prolactin cell aging and promoting prolactin secretion Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of a small molecular compound in preparing a medicament for treating or improving pituitary prolactin cell aging and promoting prolactin secretion, wherein the small molecular compound is 4-bromo-1-hydroxy-2-naphthoic acid. The small molecular compound has good central nervous system permeability, can target an ErbB4 receptor, effectively promote the expression and secretion of the pituitary prolactin cells in vitro experiments, improve the activity of the D-galactose-induced aging pituitary prolactin cells, and promote the expression and secretion of the prolactin. In vivo experiments, the small molecular compound can effectively improve the secretion of prolactin of mice with aging models, and is expected to become a candidate therapeutic drug for treating hypopituitarism and nervous system degenerative diseases related to hypopituitarism and hypopituitarism secretion.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of a small molecular compound in preparation of a medicine for improving pituitary prolactin cell aging and promoting prolactin secretion.
Background
Aging and related diseases place a heavy burden on the elderly population and their relatives and society due to the prolongation of human life and the aggravation of aging population, wherein pituitary aging is one of the important factors affecting the quality of life of the elderly.
The pituitary is the main regulating organ of human body homeostasis, and reacts dynamically to the changing hormone metabolism environment. However, with age, imbalance in the response of the aging pituitary to endogenous and exogenous signals can lead to abnormal secretion of Prolactin (PRL), often associated with hypopituitarism and the development of aging-related parkinson's disease and type II diabetes, among others. At the cellular molecular level, cell senescence is one of the important causes of decreased levels of pituitary prolactin. Although the research on cell senescence and senescence-associated diseases has been rapidly progressed in recent years, there is a distance between the transformation of results and clinical application, so it is of great importance to find a safe and effective medicament against pituitary prolactin cell senescence.
ErbB4 (receptor epidermal growth factor) belongs to the family of receptor tyrosine kinases, members of which include ErbB1-ErbB4.ErbB receptors can be activated by a variety of EGF family proteins and play an important role in the development of the nervous system and in the development of related neurodegenerative diseases. ErbB4 is capable of interacting with neuromodulation proteins, such as Nrg1, to produce intracellular signaling and play an important role in delaying the progression of aging-related diseases.
The application of small molecule compound agonists targeting ErbB4 receptors in pituitary prolactin cell aging and promoting prolactin secretion has not been reported and patented yet.
Disclosure of Invention
[ Technical problem ]
The invention aims to provide an application of a small molecular compound 4-bromo-1-hydroxy-2-naphthoic acid (C 11H7BrO3, english full name: 4-bromo-1-hydroxyne-2-carboxilic acid) of a targeted ErbB4 receptor in preparation of anti-pituitary prolactin cell aging drugs, provides a new direction for research of the anti-pituitary cell aging drugs, solves the technical problem that the prior art lacks of preparing the anti-pituitary cell aging drugs by utilizing the small molecular compound, and provides candidate therapeutic drugs for treating hypopituitarism and related diseases based on pituitary cell aging.
Technical scheme
The invention provides an application of a small molecular compound with the following structure or pharmaceutically acceptable salt thereof in preparing an anti-pituitary lactation cell aging medicament;
In one embodiment of the invention, the pharmaceutically acceptable salts include inorganic salts and/or organic salts; the inorganic salt comprises any one or more of sodium salt, calcium salt, potassium salt, magnesium salt, silver salt and lithium salt; the organic salts include meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, terbutamine salt, and N, N-dibenzylethylenediamine salt.
In one embodiment of the invention, the small molecule compound is a small molecule compound that targets the ErbB4 receptor. The small molecule compounds are useful as agonists of the ErbB4 receptor and are capable of specifically binding to the ErbB4 receptor to exert receptor agonism.
The invention also provides a medicine for treating diseases related to hypophysis prolactin cell aging, such as hypophysis gland and the like by using the micromolecular compound.
In one embodiment of the present invention, the diseases related to aging of the pituitary prolactin cells include, in addition to hypopituitarism: parkinson's disease and type II diabetes.
In one embodiment of the invention, the small molecule compounds are used in the manufacture of a medicament for treating or ameliorating aging of pituitary prolactin cells and promoting secretion of prolactin.
In one embodiment of the invention, the medicament further comprises a pharmaceutical excipient; the pharmaceutical excipients comprise any one or more of the following: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculant and deflocculant, filter aids, and release retarders.
In one embodiment of the invention, the medicament further comprises a drug carrier; the drug carrier is selected from the group consisting of microcapsules, microspheres, nanoparticles, and liposomes.
In one embodiment of the present invention, the dosage form of the drug is selected from the group consisting of injection, freeze-dried powder for injection, suspension, implant, suppository, capsule, tablet, pill and oral liquid.
In one embodiment of the invention, the concentration of the small molecule compound ranges from 1.25 nM to 5nM.
In one embodiment of the invention, the application of the small molecular compound in preparing the medicine for treating diseases related to pituitary prolactin cell aging is characterized in that the small molecular compound has no obvious influence on the activity of the pituitary prolactin cells in the concentration range of 1.25-5nM by detecting the toxic effect of the small molecular compound on the pituitary prolactin cells.
The invention explores the protection effect of the small molecular compound on the aging of the pituitary prolactin cells in vivo and in vitro in the aging model of the pituitary prolactin cells.
The small molecular compound is obtained by establishing a targeted ErbB4 protein extracellular functional domain for virtual screening, taking an in-vitro pituitary lactating cell model as a platform, exploring the effects of the small molecular compound on improving the activity of pituitary lactating cells and promoting the expression and secretion of lactating under the condition of aging pathology, and verifying the influence of the small molecular compound on the secretion of lactating of aging mice by using a D-galactose induced mouse aging model. Experimental results show that the small molecular compound has good central nervous system permeability, can target an ErbB4 receptor, effectively promote the expression and secretion of the pituitary prolactin cells in vitro experiments, improve the activity of the D-galactose-induced aging pituitary prolactin cells, and promote the expression and secretion of the prolactin; in vivo experiments, the small molecular compound can effectively improve the secretion of the prolactin of the aging mice, and is expected to become a candidate therapeutic drug for treating hypopituitarism and nervous system degenerative diseases based on hypophysis prolactin cell aging.
[ Advantageous effects ]
The beneficial effects of the invention are mainly as follows: compared with the prior art, the small molecular compound has good central nervous system permeability as a ligand substitute, can target an ErbB4 receptor to effectively improve the aging of pituitary prolactin cells in-vitro experiments, and effectively improve the secretion of the D-galactose-induced prolactin of aging mice in-vivo experiments, and has good application prospect in treating diseases related to the deficiency of the prolactin such as hypopituitarism, parkinson disease and type II diabetes.
Drawings
FIG. 1 is a schematic representation of the effect of ErbB4 receptor small molecule compounds on rat pituitary prolactin cell viability;
FIG. 2 is a schematic representation of ErbB4 receptor small molecule compounds promoting the expression and secretion of prolactin by pituitary prolactin cells;
FIG. 3 is a schematic representation of the protective effect of ErbB4 receptor small molecule compounds on D- (+) -galactose (D-gal) induced senescent pituitary prolactin cells;
FIG. 4 is a schematic representation of anti-pituitary prolactin cell senescence promoting prolactin expression by ErbB4 receptor small molecule compounds;
FIG. 5 is a schematic representation of anti-pituitary prolactin cell senescence and promotion of prolactin secretion by ErbB4 receptor small molecule compounds;
FIG. 6 is a schematic representation of serum prolactin changes in mice of each group, control, aging model (D-gal), and D-gal+ErbB4receptor small molecule co-administered group (D-gal+E4A).
Detailed Description
Structure of ErbB4 receptor small molecule compound agonist (E4A):
Description of the sources of small molecule compounds: the small molecule compound used was C 11H7BrO3 powder, purchased from Sigma-Aldrich (Shanghai) tracking Co.Ltd. (cat# S987115).
Example 1: verification of toxicity of ErbB4 receptor Small molecule Compounds on rat pituitary prolactin cells
Specifically, the method comprises the following steps of
GH3 cells in the logarithmic growth phase were trypsinized and plated in 96-well cell culture plates (5000 cells/well), 100. Mu.L of cell suspension was plated per well, and divided into 6 groups (0 nM, 1.25nM, 2.5nM, 5nM, 10nM and 20nM small molecule compounds, respectively), each group was provided with 6 duplicate wells, and the limbal wells were filled with an equal amount of serum-free medium to prevent limbic effects.
After shaking up the cells, placing the cells in an incubator, when the cells are in a logarithmic growth phase, adding small molecule compound solutions with different concentrations into each of 5 groups, so that the concentrations reach 1.25nM, 2.5nM, 5nM, 10nM and 20nM respectively, and adding the same amount of serum-free medium into a control group.
After 48h incubation, 10. Mu.L of CCK8 solution was added to each of the wells and after incubation at 37℃for 2h, the OD of each well was measured immediately with a microplate reader at a wavelength of 450 nm.
As shown in FIG. 1, the concentration of the small molecule compound is in the range of 0-5nM, and the small molecule compound has no cytotoxicity on the activity of GH3 cells.
Example 2: verifying the effect of ErbB4 receptor small molecule compounds on the promotion of prolactin expression and secretion by pituitary prolactin cells
GH3 cells in the logarithmic growth phase were trypsinized and plated in 48-well cell culture plates (1X 10 5 cells/well), 300. Mu.L of cell suspension was plated in each well, divided into 4 groups (0 nM, 5nM, 10nM, and 20nM small molecule compounds, respectively), 6 replicates of each group, and the limbal wells were filled with equal amounts of serum-free medium to prevent edge effects.
After shaking up the cells, placing the cells in an incubator, when the cells are in a logarithmic growth phase, adding 300 mu L of small molecule compound solutions with different concentrations into each of 3 groups, so that the concentrations reach 5nM, 10nM and 20nM respectively, and adding the same amount of serum-free culture medium into a control group.
After 48 hours of cell culture, the cells and the culture medium of the experimental conditions were collected, proteins were extracted, and the expression and secretion of the PRL of GH3 cells were examined.
The results are shown in FIG. 2, and the small molecule compound treatment of 5-20nM can significantly promote GH3 prolactin expression and secretion.
Example 3: verifying the effect of ErbB4 receptor small molecule compounds on anti-pituitary prolactin cell aging and on promoting prolactin secretion
Step one:
GH3 cells in the logarithmic growth phase were taken, digested with pancreatin, inoculated into 96-well cell culture plates (5000 cells/well), 100. Mu.L of cell suspension was inoculated per well, and divided into 5 groups (blank control group, D-gal administration treatment group, small molecule compound combination D-gal administration treatment group of different concentrations, respectively), 6 duplicate wells were set per group, and the marginal wells were filled with an equal amount of serum-free culture medium to prevent marginal effects.
The cells were placed in an incubator and when the cells were in the logarithmic growth phase, 100. Mu.L of serum-free medium was added to each well of the blank group, 100. Mu.L of 100mM D-gal solution was added to each well of the D-gal administration group, and 100. Mu.L of solutions containing 2.5nM, 5nM, 10nM, 20nM small molecule compound and 100mM D-gal, respectively, were added to the combination administration group.
After 48h incubation, 10. Mu.L of CCK8 solution was added to each of the wells and after incubation at 37℃for 2h, the OD of each well was measured immediately with a microplate reader at a wavelength of 450 nm.
As shown in FIG. 3, 2.5-10nM small molecule compound treatment significantly improved D-gal-induced cell viability decline.
Step two:
GH3 cells in the logarithmic growth phase were trypsinized and plated in 48-well cell culture plates (1X 10 5 cells/well), and 300. Mu.L of cell suspension was plated in each well and divided into 4 groups (blank, D-gal-treated group, 5nM small molecule compound-D-gal-treated group, 10nM small molecule compound-D-gal-treated group) and 6 replicates of each group.
After 12 hours of cell culture, 300. Mu.L of serum-free medium was added to each well of the blank group, 300. Mu.L of a 100mM concentration of D-gal solution was added to each well of the D-gal administration group, 300. Mu.L of a 100mM mixed solution of D-gal and 5nM of a small molecule compound was added to each well of the administration group, 300. Mu.L of a 100mM mixed solution of D-gal and 10nM of a small molecule compound was added to each well of the administration group, and after further culturing for 48 hours, the cells and the culture medium of the experimental conditions were collected, proteins were extracted, and the expression levels (p 53, p21, p 16) and the expression and secretion of PRL of GH3 cell senescence-associated proteins were examined.
The results are shown in FIGS. 4-5, and the treatment with 5nM small molecule compounds significantly improved D-gal-induced cellular senescence and promoted prolactin expression and secretion.
Example 4: verification of the Effect of ErbB4 receptor Small molecule Compounds on D-gal induced secretion of prolactin in senescent mice
Experimental animals: SPF-class male C57BL/6 mice, 8 weeks old, weighing 20-25g.
(1) Experimental method
Modeling and group administration
1) Model building
Male C57BL/6 mice are adaptively raised in an animal central barrier environment for 1 week, the states of the mice are observed, and the mice are marked and randomly grouped, namely a blank control group, a ageing group and a small molecule compound administration group. The blank group was intraperitoneally injected with 0.2ml of physiological saline every day, and the other groups of mice were intraperitoneally injected with an equal volume of 100mg/kg of D-gal solution every day, and molding was continued for seven weeks.
2) Administration of small molecule compounds
Seven weeks after molding, the blank group was intraperitoneally injected with 0.2ml of a sterile PBS solution, the aging group was intraperitoneally injected with 0.2ml of a 100mg/kg D-gal solution, and the small molecule compound administration group was intraperitoneally injected with 0.2ml of a mixed solution containing 100mg/kg D-gal and a small molecule compound (400 ng/kg/D) for 4 weeks.
(2) ELISA detection of serum prolactin level in mice of each group
The collected mouse blood sample was centrifuged at 1000g for 20min, and the supernatant was collected. Blank wells, negative control wells, and sample wells were set, and each set was tested 4 times in duplicate. 100 mu L of diluent (only adding chromogenic solution and stop solution; dilution ratio is 1:1) is added into the blank hole, 100 mu L of coating solution is added into the negative control hole, the supernatant sample to be tested is diluted by the coating solution according to the ratio of 1:5, 100 mu L of sample diluent is added into the sample hole, the reaction hole is sealed by a sealing plate, and the coating is carried out at 4 ℃ for 48 hours.
After 48h of coating, washing the wells with washing liquid for 3 times and 3 min/time. 100. Mu.L of 5% bovine serum was added to each well, and the wells were blocked at room temperature for 1 hour. And filling the reaction holes with a sealing liquid during sealing, removing bubbles in the holes, and washing the holes with a washing liquid for 3 times after sealing, wherein each time is 3min.
The rabbit anti-PRL antibody was diluted with 5% bovine serum (dilution ratio: 1:500), 100. Mu.L of PRL antibody dilution was added to each well, the wells were sealed with a sealing plate, and incubated at 4℃for 48h. After the incubation was completed, the cells were washed 3 times with a washing solution for 3 min/time.
Horseradish peroxidase (HRP) -labeled goat anti-rabbit antibody was diluted with 5% bovine serum (dilution ratio: 1:500), 100. Mu.L of this antibody dilution was added to each well, and incubated for 2h at 37 ℃. After the incubation was completed, the cells were washed 3 times with a washing solution for 3 min/time. 100. Mu.L of substrate chromogenic solution is added to each well, incubated at 37℃for 2 hours in the absence of light, 100. Mu.L of stop solution is added to stop the reaction, and the OD value of each well at 450nm is immediately detected using a microplate reader.
As shown in fig. 6, serum prolactin of mice in the aging group was significantly lower than that of mice in the control group (demonstrating successful modeling of the hypophysis model), and serum prolactin levels of mice were significantly increased after treatment with the small molecule compound, indicating that the D-gal modeling induced aging mice had significantly reduced prolactin secretion levels, and that administration of the small molecule compound treatment significantly improved the decline in prolactin secretion in the aging mice.
From the results, it can be seen that the targeted ErbB4 small molecule compound has good research prospect for preparing the medicament for resisting pituitary prolactin cell aging and promoting prolactin secretion.
The above examples are not intended to limit the scope of the invention nor the order of execution of the steps described. The present invention is obviously modified by a person skilled in the art in combination with the prior common general knowledge, and falls within the scope of protection defined by the claims of the present invention.
Claims (10)
1. Application of a small molecular compound with the following structure or pharmaceutically acceptable salt thereof in preparing anti-pituitary lactalbumin cell senescence medicaments;
2. The use according to claim 1, wherein the pharmaceutically acceptable salts comprise inorganic salts and/or organic salts; the inorganic salt comprises any one or more of sodium salt, calcium salt, potassium salt, magnesium salt, silver salt and lithium salt; the organic salts include meglumine salt, tromethamine salt, diethylamine salt, lysine salt, choline salt, arginine salt, terbutamine salt, and N, N-dibenzylethylenediamine salt.
3. The use according to claim 1, wherein the small molecule compound acts as an agonist of the ErbB4 receptor and is capable of specifically binding to the ErbB4 receptor to exert a receptor agonism.
4. Use of a small molecule compound of the structure shown in claim 1 for the manufacture of a medicament for the treatment of diseases related to aging of pituitary prolactin cells.
5. Use of a small molecule compound of the structure shown in claim 1 for the manufacture of a medicament for the treatment of hypopituitarism.
6. The use according to claim 4, wherein the diseases associated with aging of the pituitary prolactin cells comprise: type II diabetes.
7. A small molecule compound of the structure shown in claim 1 for use in the manufacture of a medicament for treating or ameliorating aging of pituitary prolactin cells and promoting secretion of prolactin.
8. The use according to any one of claims 1 to 7, wherein the medicament further comprises a pharmaceutical excipient; the pharmaceutical excipients comprise any one or more of the following: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, flocculant and deflocculant, filter aids, and release retarders.
9. The use according to any one of claims 1 to 7, wherein the medicament further comprises a medicament carrier; the drug carrier is selected from the group consisting of microcapsules, microspheres, nanoparticles, and liposomes.
10. The use according to any one of claims 1 to 7, wherein the pharmaceutical dosage form is selected from the group consisting of injection solutions, freeze-dried powder for injection, suspensions, implants, embolization agents, capsules, tablets, pills and oral liquids.
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