Application of 11 beta-HSD 1inhibitor in protecting neural stem cells in stress environment
阅读说明：本技术 11β-HSD1抑制剂在应激环境下保护神经干细胞的应用 (Application of 11 beta-HSD 1inhibitor in protecting neural stem cells in stress environment ) 是由 林函 连庆泉 马君梅 李军 刘华程 张旭彤 于 2020-06-11 设计创作，主要内容包括：本发明提供了11β-HSD1抑制剂用于制备神经保护剂,特别是应激环境下神经干细胞保护剂以及用于制备促进神经干细胞增殖和迁移的药物的用途。本发明首次证实神经干细胞上存在糖皮质激素代谢酶11β-HSD1,并且发现11β-HSD1抑制剂,例如BVT-14225、BVT-2733、AZD4017、AZD8329、姜黄素及类似物、以及它们的药学上可接受的盐或酯等,通过抑制神经干细胞上11β-HSD1酶的还原活性,可促进应激环境下神经干细胞的发育,降低糖皮质激素浓度,控制应激水平,为围术期应激或社会应激情况下的神经保护提供了一条新的途径。(The invention provides application of an 11 beta-HSD 1inhibitor in preparing a neuroprotective agent, in particular a neuroprotective agent under a stress environment and a medicament for promoting proliferation and migration of neural stem cells. The invention proves that glucocorticoid metabolizing enzyme 11 beta-HSD 1 exists on the neural stem cell for the first time, and discovers that 11 beta-HSD 1 inhibitors, such as BVT-14225, BVT-2733, AZD4017, AZD8329, curcumin, analogues, pharmaceutically acceptable salts or esters thereof and the like can promote the development of the neural stem cell under a stress environment, reduce the glucocorticoid concentration and control the stress level by inhibiting the reducing activity of 11 beta-HSD 1 enzyme on the neural stem cell, thereby providing a new way for neuroprotection under perioperative stress or social stress.)
Use of an inhibitor of 11 β -HSD1 for the preparation of a neuroprotective agent.
2. Use according to claim 1, wherein the 11 β -HSD 1inhibitor is selected from BVT-14225 and pharmaceutically acceptable salts thereof, BVT-2733 and its free base salts with other pharmaceutically acceptable acids, AZD4017 and its pharmaceutically acceptable salts or esters, AZD8329 and its pharmaceutically acceptable salts or esters, curcumin and analogues.
3. Use according to claim 1 or 2, wherein the neuroprotective agent is a neuroprotective agent against neural stem cells in a stressful environment.
4. Use according to any one of claims 1 to 3, wherein the stress environment comprises acute and chronic stress environments of the following diseases or operations:
1) fetal surgery, such as that performed by a fetal spina bifida, twins syndrome, prevascular, congenital diaphragmatic hernia;
2) intrapartum surgery or neonatal surgery, such as intrapartum surgery or neonatal surgery due to a herniated navel, congenital double aortic arch heart disease, esophageal atresia, esophageal tracheal fistula, hiatal hernia;
3) chronic social stress environments, such as maternal-fetal segregation during pregnancy, neonatal period, violent experiences, and chronic stress environments caused by improper home care.
Use of an inhibitor of 11 β -HSD1 for the manufacture of a medicament for promoting proliferation and migration of neural stem cells.
6. Use according to claim 5, wherein the 11 β -HSD 1inhibitor is selected from BVT-14225 and a pharmaceutically acceptable salt thereof, BVT-2733 and a free base thereof and a salt of a free base thereof with a pharmaceutically acceptable acid, AZD4017 and a pharmaceutically acceptable salt or ester thereof, AZD8329 and a pharmaceutically acceptable salt or ester thereof, curcumin and analogues thereof.
7. The use according to claim 5 or 6, wherein the medicament for promoting proliferation and migration of neural stem cells can be used for the prevention and/or treatment of the following diseases or conditions: stroke, alzheimer's disease, parkinson's syndrome, craniocerebral trauma sequelae; can also be used as an adjuvant before, during or after the following surgery: fetal surgery (e.g., fetal surgery due to a fetal spina bifida, twins syndrome, prevascular, congenital diaphragmatic hernia), intrapartum surgery, or neonatal surgery (e.g., intrapartum surgery due to a bulgy, congenital double aortic arch heart disease, esophageal atresia, esophageal tracheal fistula, hiatal hernia, or neonatal surgery).
8. The use according to any one of claims 2-4 and 6-7, wherein the analogue of curcumin is the following compound:
the structural formulas of the curcumin analogues A01-A17, B01-B17 and C01-C17 are shown in the following tables 2-4:
TABLE 2
TABLE 3
TABLE 4
The structural formula of curcumin analogs 2-16 is shown in table 5 below:
TABLE 5
Technical Field
The invention belongs to the field of neuroprotection and stress research, and particularly relates to application of an 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD 1) inhibitor (such as BVT-14225) in preparation of a neural stem cell protective agent and in preparation of a medicine for promoting proliferation and migration of neural stem cells.
Background
Neural Stem Cells (NSCs) are pluripotent stem cells of the central nervous system, have the proliferation capacity of continuous self-renewal, have the differentiation capacity towards neurons, astrocytes and oligodendrocytes and the migration capacity towards specific brain regions, and have an important role in maintaining the homeostasis of the central nervous system and the development of normal brain tissues. In addition, NSC development plays an important role in repairing damaged nerve cells and maintaining cognitive function. The fetal and neonatal brains contain a large number of neural stem cells. At present, preclinical research or clinical test of neural stem cell transplantation for treating nerve injury diseases is carried out, and the curative effect is obvious, wherein the diseases comprise diseases such as apoplexy, Alzheimer disease, Parkinson syndrome, craniocerebral trauma sequelae and the like.
The stress environment referred to in the present invention is an acute stress environment such as surgery or a chronic social stress environment. The acute stress environment related to the development of the neural stem cells specifically refers to maternal operation in pregnancy, fetal operation in pregnancy, neonatal operation or neural stem cell transplantation operation and the like. The chronic social stress environment related to the development of the neural stem cells specifically refers to maternal-fetal segregation, violent experience, improper home care and the like in the pregnancy and the newborn period.
Glucocorticoid is an extremely important regulatory molecule in the body, plays an important role in regulating development, growth, metabolism, immune function and the like of the body, is the most important regulatory hormone of stress response of the body, and is also the most widely and effectively anti-inflammatory and immunosuppressive agent used clinically. As an important stress hormone, glucocorticoids are significantly elevated perioperatively in pregnant women, fetuses and infants, and also in cases of chronic social stress. Elevated glucocorticoids, or large or prolonged doses of glucocorticoids, can have some adverse effects on the development of the central nervous system, particularly NSCs. In the in vitro culture of NSCs, active glucocorticoid (CORT) significantly inhibits the proliferation of neural stem cells; the high concentration of cortisol can inhibit the differentiation of the neural stem cell line (HPC03A/07) to neurons, but does not affect the differentiation to astrocytes; in a pregnant mouse constraint stress model, the migration capacity of neural stem cells of offspring pups is also obviously reduced, so that the stress level of CORT influences the neural development. Water maze experiments of an early stress model of a fetal rat show that compared with a control group, the latency period and the total track distance of SD rats in the stress group are obviously prolonged in acquired training. Thus, the stress level of CORT can impair cognitive function.
11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-Hydroxysteroid dehydrogenase type 1, 11 beta-HSD 1) is a glucocorticoid metabolizing enzyme and has the function of catalyzing the interconversion of inactive cortisone (11-Dehydrocorticosterone, DHC, in rodents) and active hydrocortisone (CorT, in rodents), thereby maintaining the stable local tissue active glucocorticoid concentration. The 11 beta-HSD 1inhibitor comprises BVT and AZD series, wherein the BVT series comprises BVT-14225 and BVT-2733, and the AZD series comprises AZD4017 and AZD8329, which have pharmacological action of inhibiting 11 beta-HSD 1 activity; in addition, curcumin and its analogs also have the effect of inhibiting 11 β -HSD1 activity.
Disclosure of Invention
The inventor establishes a stress model of rat primary neural stem cells by using DHC, and identifies the primary cells by immunofluorescence staining of a neural stem cell marker Nestin (Nestin) and Sex-determining gene-related transcription factor-2 (Sex determining region Y-transcription factor 2, Sox 2). Then extracting Ribonucleic acid (RNA) of the neural stem cell, and carrying out reverse transcription Polymerase Chain Reaction (PCR), common amplification PCR and nucleic acid gel electrophoresis experiments to identify 11 beta-HSD 1 on the neural stem cell.
The research of the inventor is divided into two parts, the first part uses an EdU experiment to detect the influence of CORT and DHC on the proliferation capacity of the neural stem cells, and the concentration of DHC under stress is determined. A second set of experiments, followed at this concentration, investigated the effect of BVT on neural stem cell development by modulating 11 β -HSD1 enzymatic activity.
The inventors tested the cytotoxicity of DHC and BVT using Lactate Dehydrogenase (LDH) release assay; detecting the influence of BVT on the activity of 11 beta-HSD 1 by adopting an Ultra performance liquid chromatography-mass spectrometry (UPLC-MS); detecting the influence of DHC and BVT on the expression level of 11 beta-HSD 1 protein of the neural stem cells by adopting a western blot experiment (Westernblot, WB); detecting the influence of DHC and BVT on the proliferation capacity of the neural stem cells by adopting an EdU experiment; the influence of DHC and BVT on the differentiation capacity of the neural stem cells is detected by adopting an immunofluorescence experiment of a neuron marker-neuron nuclear antigen antibody (NeuN) and an astrocyte marker-Glial Fibrillary Acidic Protein (GFAP); the effect of DHC and BVT on the migratory capacity of neural stem cells was examined using the scratch test.
Based on the above experimental studies and research results, the inventors have completed the present invention.
The present invention provides the use of an inhibitor of 11 β -HSD1 for the preparation of a neuroprotective agent.
According to the invention, the 11 beta-HSD 1inhibitor is selected from BVT-14225(CAS registry number 376638-65-2) and pharmaceutically acceptable salts thereof, BVT-2733(CAS registry number 376641-65-5) and free base thereof and salt thereof with other pharmaceutically acceptable acid, AZD4017(CAS registry number 1024033-43-9) and pharmaceutically acceptable salt or ester thereof, AZD8329(CAS registry number 1048-70-7) and pharmaceutically acceptable salt or ester thereof, curcumin (CAS registry number 458-37-7) and analogues thereof, and the like. Wherein the curcumin analogues may be compounds A01-A17, B01-B17 and C01-C17 described in Han Lin et al, "Mono-carbonyl curcumin analogues as 11 beta-hydro-specified dehydrogenase1inhibitor, Bioorganic & Medicinal chemistry letters, Vol.23, No. 15, No. 8 2013, p 4362-4366), in particular compounds A02, A06, A10, B02, B06, B13, B14, C02, C06 and C13; and Curcumin analogs 2-16 described in the inventors' article (Guo-Xin Hu, Han Lin et al, Current as apotent and selective inhibitor of 11 β -hydrosteroid hydrogene 1: stimulating lipid in high-fat-diet-derived rates, PLOSONE, Vol.8, No. 3, 3 months 2013, e 49976).
The 11 β -HSD 1inhibitor has the structure shown in Table 1 below:
TABLE 1
The structural formulas of the curcumin analogues A01-A17, B01-B17 and C01-C17 are shown in the following tables 2-4:
TABLE 2
TABLE 3
TABLE 4
The structural formula of curcumin analogs 2-16 is shown in table 5 below:
TABLE 5
The pharmaceutically acceptable salts mentioned in the present invention may be acid addition salts or base addition salts depending on the structure of the compound. For example, acid addition salts with the following inorganic or organic acids may be mentioned: hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, formic acid, acetic acid, tartaric acid, fumaric acid, citric acid, malic acid, oxalic acid, ascorbic acid, succinic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, etc., or may be a salt with sodium ion, potassium ion, calcium ion, ammonium ion, etc.
The pharmaceutically acceptable ester referred to in the present invention means an ester of a compound having a carboxyl group or a hydroxyl group with an alcohol compound, an amino compound, or a carboxyl compound, and may be, for example, a carbonate or a carbamate.
According to the invention, the neuroprotective agent is a neuroprotective agent for neural stem cells in a stressful environment.
According to the present invention, the stress environment includes, but is not limited to, acute stress environment and chronic stress environment such as following diseases or operations:
1) fetal surgery: fetal spina bifida, twins transfusion syndrome, anterior vessels, congenital diaphragmatic hernia, and the like;
2) intrapartum surgery or neonatal surgery: umbilicus bulging, congenital double aortic arch heart disease, esophageal atresia, esophageal tracheal fistula, hiatal hernia, etc.;
3) chronic social stress environments include, but are not limited to, the following: maternal and infant segregation during pregnancy and neonatal period, violent experience, improper home care and the like.
The invention also provides the use of an inhibitor of 11 β -HSD1 for the manufacture of a medicament for promoting the proliferation and migration of neural stem cells.
According to the present invention, the agent for promoting the proliferation and migration of neural stem cells can be used for the prevention and/or treatment of the following diseases or conditions: stroke, alzheimer's disease, parkinson's syndrome, craniocerebral trauma sequelae; can also be used as an adjuvant before, during or after the following surgery: fetal surgery (e.g., fetal surgery due to a fetal spina bifida, twins transfusion syndrome, prevascular, congenital diaphragmatic hernia, etc.), intrapartum surgery, or neonatal surgery (e.g., intrapartum surgery or neonatal surgery due to a bulgy, congenital double aortic arch heart disease, esophageal atresia, esophageal tracheal fistula, hiatal hernia, etc.).
The research related to the invention proves that 11 beta-HSD 1 serving as glucocorticoid metabolizing enzyme exists on the neural stem cells for the first time, and the 11 beta-HSD 1 inhibitors, such as BVT-14225 (which is currently used as a medicament for treating diabetes), BVT-2733, AZD4017, AZD8329, curcumin, analogues thereof, pharmaceutically acceptable salts or esters thereof and the like, are found to promote the development of the neural stem cells under a stress environment, reduce the glucocorticoid concentration, control the stress level and provide a new way for neuroprotection under perioperative stress or social stress conditions by inhibiting the reducing activity of 11 beta-HSD 1 enzyme on the neural stem cells.
Drawings
Fig. 1 shows an inverted fluorescence microscope photograph of multicellular neurospheres formed by proliferation of primary neural stem cells.
FIG. 2 shows the photograph of the immunofluorescent staining of Nestin with neurospheres (A) and a monolayer of neural stem cells (B), with 4 ', 6-Diamidino-2-phenylindole (4', 6-Diamidino-2-phenylindole, DIPA) counterstaining of nuclei, and merge being the overlap of the immunofluorescent signals of Nestin and DIPA at the same position.
Figure 3 shows a photograph of Sox2 immunofluorescent staining of neurospheres (a) and monolayers of neural stem cells (B), DIPA counterstained nuclei, merge is the overlap of the Sox2 and DIPA immunofluorescent signals at the same location.
FIG. 4 shows the results of electrophoretic identification of neural stem cells expressing 11 β -HSD 1.
Figure 5 shows an immunofluorescence photograph of a neural stem cell proliferation assay. EdU represents proliferating nuclei, DIPA counterstains all nuclei, and merge is the overlap of EdU and DIPA immunofluorescence signals at the same position.
FIG. 6 shows WB bands expressed by neural stem cell 11 β -HSD1 protein. beta-Tubulin (beta-Tubulin) is used as an internal reference protein.
Figure 7 shows an immunofluorescence photograph of a neural stem cell proliferation assay. EdU represents proliferating nuclei, DIPA counterstains all nuclei, and merge is the overlap of EdU and DIPA immunofluorescence signals at the same position.
Figure 8 shows an immunofluorescence photograph of a neural stem cell differentiation experiment. NeuN stands for neurons, GFAP stands for astrocytes, DIPA counterstains all nuclei, merge is the overlap of EDU and DIPA immunofluorescence signals at the same location.
Fig. 9 shows a photograph of a neural stem cell scratch experiment.
Detailed Description
Hereinafter, the use of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. The BVT used in the examples below is BVT-14225.
EXAMPLE 1 identification of Primary neural Stem cells
After 5-7 days of culture, the primary neural stem cells can gradually proliferate to form multicellular spheroids with good refractivity (as shown in figure 1), and neurospheres of 400-.
The primary cells extracted by the immunofluorescence staining method are subjected to immunofluorescence staining of neural stem cell markers Nestin and Sox2 (shown in attached figures 2 and 3), and the results show that the proportion of Nestin and Sox2 positive cells can reach more than 96%. The cultured primary cells were demonstrated to be neural stem cells.
Except that the culture plate before the inoculation of the scratch experiment cells does not need the matrigel pretreatment, other experiment cell culture plates are pretreated by the matrigel and are placed in a cell culture box for 6-12 hours so as to promote the adherent growth of the neural stem cells.
Example 2 identification of 11 β -HSD1 on neural Stem cells
Extracting RNA of neural stem cells, performing reverse transcription PCR, common amplification PCR and nucleic acid gel electrophoresis experiments to identify 11 beta-HSD 1 (shown in figure 4) on the neural stem cells, and taking housekeeping gene RS16 as internal reference. The results indicated that 11 β -HSD1 was expressed on neural stem cells.
Example 3 stress modeling with CORT to determine the toxic concentration of DHC under stress
The neural stem cells were randomly divided into 6 groups, namely, a normal control group (group C), a solvent control group (group D), a CORT group, a 0.1. mu. MDHC group (group DHC O.1), a 1. mu.M DHC group (group DHC 1.0), and a 10. mu.M DHC group (group DHC 10), and the NSC was administered at a rate of 0.5 × 10 per well5The cells were inoculated in 24-well plates previously loaded with a microplate at a density of 500. mu.L/well, treated for 48 hours, and then 5-ethynyl-2' deoxyuridine (EdU) was added to the cells at a final concentration of 10. mu.M 4 hours before the end of the experiment, and passed through a Click-iT cellTMImmunofluorescence experiments were performed with the EdUAlexa Fluor488 kit instructions (see Table 6 for reagent formulations). The proliferation capacity of NSC was represented by counting the EdU + cells and total cells by using an upright Leica microscope (see FIG. 5) and calculating the proliferation rate by ImageProPlus 6.0 software. Growth rate (%) ═ EdU+× 100% of total cells.
TABLE 6 EdU reaction liquid composition and ratio
The results show that the NSC proliferation capacities of the group C, the group D, the group DHC 0.1 and the group DHC 1.0 are not statistically different; the proliferation potency of NSC was significantly inhibited in both CORT and DHC10 groups compared to D group (P <0.05), while the proliferation potency of neural stem cells was not statistically different in CORT and DHC10 groups (P >0.05) (see Table 7). The above results show that 1. mu.M CORT and 10. mu.M DHC have the same degree of inhibition effect on the proliferation of neural stem cells, and the stress environment is established by using 10. mu.M DHC as the molding concentration in all the experiments.
TABLE 7 Effect of CORT and DHC on the proliferative capacity of neural stem cells (S) ((S))n＝3)
P <0.05 compared to group C; compared to group D, # P < 0.05; compared to the CORT group, $ P > 0.05.
EXAMPLE 4 LDH assay to detect BVT (i.e., BVT-14225) and DHC cytotoxicity
Neural stem cells were randomly divided into 5 groups, normal control group (group C), solvent control group (group D), 10. mu.M DHC group (group DHC), 10. mu.M BVT group (group BVT), and 10. mu.M DHC + 10. mu.M BVT group (group BD, BVT treatment started prior to DHC1 h), at 1.0 × 10 per well5The density of/mL was inoculated in 96-well plates, 100 μ L of medium was added to each well, LDH release experiments were performed after 48h of treatment according to the CytoTox96 nonradioactive cytotoxicity kit instructions, absorbance (OD490) was measured with a Bio-tek microplate reader and cell death rate was calculated to represent the cytotoxic effect of the drug on NSCs for each treatment group, cytotoxicity (%) — LDH release (OD 490)/maximum LDH release (OD490) × 100% for the treated wells.
The CytoTox96 kit comprises the following reagents: substrate Mix (Substrate Mix), Assay Buffer (Assay Buffer), LDH Positive Control (LDH Positive Control), Lysis Solution (10X) (Lysis Solution (10X)), Stop Solution (Stop Solution).
The results showed that there was no significant change in LDH release in groups C, D, DHC, BVT and BD (see Table 8), and there was no statistical significance between groups (P > 0.05). I.e. neither BVT nor DHC increased cell mortality.
TABLE 8 Effect of BVT and DHC on LDH Release from neural Stem cells: (
EXAMPLE 5 ultra high Performance liquid chromatography-Mass Spectrometry (UPLC-MS) assay for detecting 11 β -HSD1 enzymatic Activity
Neural stem cells were randomly grouped according to example 4, and NSCs were grouped at 0.5 × 10 per well5The cells were inoculated in 24-well plates with a density of/mL, 500uL of proliferation medium was added to each well, and after 48h of treatment, the cell culture medium of each group was collected and subjected to UPLC-MS treatment as follows and the concentrations of DHC and BVT in the culture medium were calculated.
Preparing CORT (0.01, 0.02, 0.1, 0.5, 1, 2 and 5 mu M) and DHC (0.03, 0.06, 0.3, 1.5, 3, 6 and 15 mu M) (standard) stock solutions with concentration gradients and 0.2 mu M Hydrocortisone (internal standard), putting 90 mu L of blank culture medium and 10 mu L of each gradient standard stock solution into a 1.5mL plastic centrifuge tube, adding 10 mu L of the internal standard, vortexing for 2min, uniformly mixing, then adding 300 mu L of methanol precipitated protein, vortexing for 3min, then centrifuging for 10min at 4 ℃ and 12000rpm, taking 100 mu L of supernatant, carrying out UPLC-MS analysis in a vial, and carrying out weighted linear regression by using the peak area ratio of the standard and the internal standard and the corresponding concentration to respectively obtain standard curve equations of CORT and DHC. Then 100 μ L of cell culture medium and 100 μ L of blank culture medium were taken to perform the same operation as above, and then the peak area ratio of the sample to the internal standard was substituted into the standard curve equation to obtain the concentrations of CORT and DHC in each culture medium sample, respectively, and the conversion rate (enzyme activity) was calculated. Enzymatic activity ═ conc (cort)/conc (dhc) × 100%.
As only the cell culture media of the DHC group and the BD group contain exogenous DHC in the experiment, and simultaneously, the mass spectrum experiment also proves that the C group, the D group and the BVT group do not contain DHC, the experiment only carries out statistical analysis on the DHC and the BD group.
The results show that the BD group conversion (enzyme activity) is significantly lower than the DHC group (P <0.01) (see table 9). Thus, BVT has a remarkable inhibitory effect on the reduction activity of 11-HSD 1.
TABLE 9 Effect of BVT on enzymatic Activity of neural Stem cell 11-HSD1 ((n＝4)
Compared to the DHC group, Δ P <0.05
EXAMPLE 6WB assay to detect the expression level of 11 β -HSD1 protein
Neural stem cell cells were randomly grouped according to example 4 at 5 × 10 per well5The concentration of each protein is measured by a BCA working kit, the protein sample amount of each lane is 25 mu g, after electrophoresis and membrane rotation, the lane is sealed by 5% skimmed milk powder for 1h, an endoproteinewhite rabbit β -Tubulin antibody (dilution ratio 1:2000) and a target protein rabbit 11 β -HSD1 antibody (dilution ratio 1:1000) are added, the lane is incubated for 18-20h at 4 ℃ in a shaking table, a goat anti-rabbit biotin secondary antibody with dilution ratio 1:5000 is added, the lane is incubated for 1h at room temperature, the lane is fully washed by TBST, the Integrated absorbance (Integrated absorbance, 48311-8663) of a target protein band and the internal reference protein band is analyzed by exposure band display (shown in figure 6), Image software J analyzes Integrated absorbance (Integrated absorbance, attune) of the target protein band and the endogenous reference protein band, and HSD protein band (HSD-3611-8663)/expression level (HSD IA-3611-8678).
The BCA kit includes the following reagents: fetal Bovine Serum (BSA) stock solution, reaction solution A and Cu reagent.
The results showed no significant change in the expression level of 11 β -HSD1 protein in group C, group D, group DHC, group BVT and group BD (see Table 10), with no statistical significance between groups (P > 0.05). That is, BVT did not alter 11 β -HSD1 protein expression levels of neural stem cells.
TABLE 10 Effect of BVT on neural Stem cell 11 β -HSD1 protein expression ((
Example 7 Effect of BVT-Regulation of 11 β -HSD1 enzymatic Activity on the proliferative Capacity of neural Stem cells
Neural stem cells were randomly grouped according to example 4, and after 48h of treatment, experiments were performed according to example 3 (see fig. 7).
The result shows that the proliferation capacity of the neural stem cells of the DHC group is obviously reduced compared with that of the D group (P <0.01), and the proliferation capacity of the neural stem cells of the BVT group and the BD group has no statistical significance (P > 0.05). Compared with the DHC group, the BD group has obviously enhanced proliferation capacity of the neural stem cells (P <0.05) (see Table 11), which indicates that the BVT has the function of promoting the proliferation of the neural stem cells under the stress condition.
TABLE 11 Effect of BVT on the proliferative Capacity of neural Stem cells: (
P <0.05 compared to group C; compared with group D, # P < 0.05; Δ P <0.05 compared to DHC group.
Example 7 Effect of BVT on the ability of 11 β -HSD1 to modulate enzymatic Activity on neural Stem cell differentiation
Neural stem cells were randomly grouped according to example 4, and NSCs were 1.0 × 105The cells were seeded in a 24-well plate containing a microplate in a density of 500 uL/well, and cultured in a cell culture box for 24 hours, then replaced with a differentiation medium, DMEM/F12 (3: 1) containing a mixture of 1% N-2 additive (100X) and 1% streptomycin, and the drug treatment was terminated after 48 hours by replacing the differentiation medium. The differentiation medium was changed every two days thereafter until the total differentiation time of the cells reached 7 d. Then, the cells of each group were subjected toIn the immunofluorescent staining experiment of GFAP, NeuN and DAPI, photographs were taken with an upright leycan microscope (see fig. 8), and the ImageJ 1.51 software and the ImageProPlus 6.0 software counted the number of GFAP +, NeuN + cells and total cells, respectively, and the differentiation rates of GFAP and NeuN were calculated, respectively, to represent the astrocyte and neuron differentiation abilities of NSC, the GFAP differentiation rate (%) (the number of GFAP + cells/total cell count of × 100%, and the NeuN differentiation rate (%) (the number of NeuN + cells/total cell count of × 100%).
The results showed NeuN for group C, group D, DHC, BVT and group BD+、GFAP+The proportion of cells is hardly changed, and the difference between groups is not statistically significant (P)>0.05) (see table 12).
TABLE 12 Effect of BVT on neural Stem cell differentiation Capacity: (
Example 8 Effect of BVT-Regulation of 11 β -HSD1 enzymatic Activity on neural Stem cell migration
Neural stem cells were randomly grouped according to example 4 at 2.5 × 10 per well5The cells were seeded in a 24-well plate at a density of/mL, 500 μ L of proliferation medium was added to each well, the cells were cultured in a cell culture chamber until the bottom of the culture well was completely covered with the cells, the cells of all the experimental wells were subjected to scratch treatment using a 200 μ L sterile tip and the differentiation medium was replaced, a photograph of the scratch (0h) of the cells at that time was immediately taken with an inverted leica microscope (see fig. 9), then the cells were processed for 48h in groups, and then a photograph of the scratch (48h) at that time was taken at the 48h photographing site was repeated (see fig. 9). ImageJ 1.51 software calculated the "healing" rate of the scratch pitch before and after the treatment, thereby representing the migration ability of nsc.mobility (%) (scratch pitch area of 0 h-scratch pitch area of 48 h)/scratch pitch area of 0h × 100% of 0 h.
The results show that the migration capacity of the neural stem cells in the DHC group is slightly reduced compared with that in the D group, but the migration capacity is not statistically significant (P > 0.05); compared with the DHC group, the BD group has a remarkably enhanced migration capability of the neural stem cells (P <0.05) (see Table 13), which indicates that the BVT has the function of promoting the migration of the neural stem cells under the stress condition.
TABLE 13 Effect of BVT on migration ability of neural Stem cells: (n＝3)
Compared to the DHC group, Δ P <0.05
And (4) conclusion:
the results of the above studies demonstrated the presence of 11 β -HSD1 on neural stem cells. It was also demonstrated that under the conditions of this experiment, inactive glucocorticoid (DHC) inhibits proliferation of neural stem cells, with no effect on differentiation and migration; BVT (BVT-14225) promotes the proliferation and migration of neural stem cells in a stress environment by inhibiting the reduction activity of 11 beta-HSD 1 enzyme, but has no influence on differentiation; and BVT has no cytotoxicity effect and does not influence the expression level of 11 beta-HSD 1 protein.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.