CN109608425B

CN109608425B - Application of compound and hydrate, salt or derivative thereof

Info

Publication number: CN109608425B
Application number: CN201811494627.1A
Authority: CN
Inventors: 谭锐; 谭立伟; 江羽
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2018-12-07
Filing date: 2018-12-07
Publication date: 2022-03-25
Anticipated expiration: 2038-12-07
Also published as: CN109608425A

Abstract

The invention relates to the field of pharmacy, in particular to application of a compound or pharmaceutically acceptable hydrate, salt or derivative thereof in inducing stem cells to be differentiated into nerve-like cells as an inducer. After the induction agent induces the bMSCs to differentiate into nerve-like cells in vitro, the bMSCs have better brain targeting property, can be more distributed at the focus of the brain, and can play a more effective treatment role.

Description

Application of compound and hydrate, salt or derivative thereof

Technical Field

The invention relates to the field of pharmacy, in particular to a compound and application of a hydrate or a salt thereof.

Background

Ischemic stroke is still one of the major diseases damaging human lives, the morbidity, disability rate and mortality rate of the ischemic stroke are extremely high, and an effective treatment and prognosis means is still lacked to date due to the complexity of the disease and the difficulty of nerve repair. A plurality of treatment schemes such as anti-inflammatory and anti-oxidation are reported at present, but no significant breakthrough is made in the evaluation of clinical treatment effect. It is appreciated that the stem cells become a new research hotspot for treating cardiovascular and cerebrovascular diseases, and also provide a new direction for treating cerebral ischemia. Among them, Bone Mesenchymal Stem Cells (bMSCs) are non-hematopoietic Stem Cells derived from Bone Marrow stromal systems, have strong proliferation ability and multi-directional differentiation potential, can differentiate into neural Cells (such as neuronal Cells, astrocytes, etc.) under the induction of specific physicochemical environment or some cytokines, and are easily obtained, easily cultured in vitro, and have strong proliferation ability, thus being commonly used for Stem cell differentiation and therapeutic research. The substances most commonly used to induce the differentiation of bMSCs into neural-like cells at present are beta-mercaptoethanol and basic fibroblast growth factor (bFGF), wherein bFGF is low in source and expensive, while beta-mercaptoethanol is liable to cause cell death and has a short maintenance time after successful induction, so that further development of novel drug molecules for stem cell therapy is required.

Disclosure of Invention

At present, there is no report on the differentiation of stem cells into neural-like cells by using the compound having the structural formula of formula (I) as an inducer, wherein the structural formula of the compound having the structural formula of formula (I) is as follows:

furthermore, the report about inducing the bone marrow mesenchymal stem cells to differentiate into nerve-like cells is provided.

The research shows that the pharmaceutically acceptable hydrate, salt or derivative of the compound has the same efficacy as the compound with the structural formula on inducing stem cells to differentiate into nerve-like cells.

The inducer of the present invention acts to induce differentiation of cells. In the specific experimental process of the invention, the compound of the formula (I) is found to have obvious effect of up-regulating NSE, GFAP and Nestin nerve cell protein expression quantity by using the compound as an inducer.

Wherein, the method for inducing the stem cell to differentiate into the nerve-like cell comprises the following steps: and (3) taking the purified stem cells, and then placing the stem cells into a culture solution containing an inducer to culture for more than 12 hours to obtain the neural-like cells. The stem cell is a bone marrow mesenchymal stem cell.

According to experiments, the survival rate of the cells is obviously reduced after the concentration of the inducer exceeds 10 mu M, and the survival rate of the cells is higher than 90% in the range of 2-10 mu M, so that the concentration of the inducer is selected to be 2-10 mu M. The inducer herein refers to the compound of formula (I) described above.

The compound of the formula (I) is mainly used for up-regulating NSE, GFAP and Nestin nerve cell protein expression quantity, wherein the expression quantity of the proteins GFAP and Nestin reaches a peak value 24h after induction, the expression quantity of the protein NSE reaches a maximum value 12h after induction, and therefore the induction culture time is 12-24 h.

The invention also aims to provide application of the compound shown as the formula (I) or pharmaceutically acceptable hydrate, salt or derivative thereof as an inducer in inducing bone marrow mesenchymal stem cells to differentiate into neural cells in preparation of medicines for preventing or/and treating ischemia or ischemic injury diseases.

Applicants used a widely used mouse cerebral MCAO ischemia model for evaluation. The results confirmed that the neural-like cells obtained after induction were effective in widening the range of neurological symptoms and cerebral infarction caused by ischemia-reperfusion of the middle cerebral artery of mice.

Wherein the medicament is a medicament for preventing or/and treating cerebral ischemia or cerebral ischemic injury diseases.

Furthermore, the cerebral ischemia diseases or cerebral ischemia injury diseases are ischemic stroke or cerebral ischemia reperfusion injury.

The applicant utilizes the widely used mouse cerebral MCAO ischemia model to evaluate, and finds that the neural-like cells obtained after induction are effective to the nerve function damage in the mouse brain.

The invention also provides application of the compound shown as the formula (I) or pharmaceutically acceptable hydrate, salt or derivative thereof as an inducer to induce mesenchymal stem cells to differentiate into neural-like cells in preparation of drugs for preventing or/and treating neuroprotection or repairing drugs.

The invention also provides a method for inducing the differentiation of the mesenchymal stem cells into neural-like cells by using the compound shown as the formula (I) or the pharmaceutically acceptable hydrate, salt or derivative thereof as an inducer, which comprises the following steps: and (3) taking the purified stem cells, and then placing the stem cells into a culture solution containing an inducer to culture for more than 12 hours to obtain the neural-like cells.

Wherein the concentration of the inducer is 2-10 μ M. The induction culture time is 12-24 h.

The invention has the beneficial effects that:

the compound and the pharmaceutically acceptable hydrate, salt or derivative thereof can induce mesenchymal stem cells to be differentiated into nerve-like cells, and the induced nerve-like cells not only have a remarkable effect on neuroprotection, but also have an obvious treatment effect on ischemic stroke and cerebral ischemia in perfusion injury diseases.

Second, after the bMSCs are differentiated into nerve-like cells in vitro, the bMSCs have better brain targeting property and can be distributed on the brain focus more, thereby playing a more effective treatment role.

Drawings

FIG. 1 is a graph of the cytotoxicity of costunolide on bMSCs;

FIG. 2 is a graph of morphological changes of bMSCs observed under an optical microscope, wherein the areas are A. blank group, B. induced for 4h, C. induced for 12h, and D. induced for 24 h;

fig. 3 is a staining immunofluorescence assay of bMSCs cytoskeleton, with regions a. blank, b. induced 4h, c. induced 12h, d. induced 24h, respectively;

FIG. 4 is a photograph of immunofluorescence staining of bMSCs cells NSE, GFAP and Nestin after induction, wherein the areas are A. blank, B. induced for 4h, C. induced for 12h and D. induced for 24 h;

FIG. 5 is a TTC staining pattern of post-operative animal brain tissue;

fig. 6 is a graph showing HE staining of rat brain tissue, wherein the regions are a. sham operation group, b. model group, c.msc group, and d. induction group.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments.

1 materials and methods

1.1 materials

1.1.1 reagents and instruments

Fetal bovine serum (Gibco batch number: 1908121); DMEM low-sugar medium (Hyclone, batch No: J180003); trypsin (Hyclone, batch No.: J180003); tetramethylazo salts (MTT) (Ruishi Bio Inc., lot number 180315); 2, 3, 5-Triphenyltetrazolium chloride (TTC) (Biotech, Inc., Source leaf, Lot: BCBR 5460V); 4% paraformaldehyde (Biosharp, lot # 180119); 96-well plates (Thermo, batch: 167008); hematoxylin staining kit (Wuhan Google Biotech Co., Ltd., batch number: G1005); NESTIN primary antibody (Wuhan Google Biotech Co., Ltd., lot number: GB 12137); GFAP primary antibody (Wuhan Google Biotech Co., Ltd., batch No: GB 11096); NSE-primary antibody (Wuhan Google Biotech, Inc., batch number: GB 11376-1); the secondary antibody CY3 goat anti-mouse (Wuhan Google Biotech Co., Ltd., batch No: GB 21301); a second 488 goat-anti-rabbit antibody (Wuhan Google Biotech Co., Ltd., batch No. GB 25303); secondary antibody CY3 goat-rabbit antibody (Wuhan Google Biotech Co., Ltd., batch No.: GB21303)

1.1.2 Experimental animals

40 SPF-grade adult male SD rats with the average body weight of 280-300g are used for preparing a cerebral ischemia-reperfusion animal model; SPF grade 2-4 week old male SD rats 2 with average body weight of 120-140g were used for preparing mesenchymal stem cells.

1.2 test methods

1.2.1 isolation and culture of bMSCs

Healthy male SD rats of 2-4 weeks old are selected to be killed after neck removal, and the whole body is soaked in 75% ethanol for disinfection for about 10 min. Taking out bilateral tibias and femurs under aseptic conditions, cutting off epiphyses at two ends, extracting 5mL of DMEM complete culture medium containing 15% fetal calf serum by using a 5mL injector, flushing bone marrow from one end, flushing from the other end to prepare single cell suspension, placing the single cell suspension in a 10mL culture dish, placing the culture dish in a 37 ℃ culture box with volume fraction of 5% CO2 and saturated humidity, completely replacing the liquid after 24h, replacing the liquid every 2d for 1 time, removing non-adherent cells, and performing mixing according to the proportion of 1: and (5) carrying out passage at a ratio of 2. Observing the growth condition of the cells every day, and when the fusion of the primary cells reaches 80% -90%, performing the following steps of 1: 2 ratio and the medium was changed to DMEM complete medium containing 10% fetal bovine serum. And (5) gradually purifying the mesenchymal stem cells through multiple passage amplification culture.

1.2.2 toxicity detection of bMSCs by Costunolide

Toxicity of costunolide on bMSCs was examined by MTT. Taking the third generation bMSCs, and arranging each hole according to 1.0 multiplied by 10⁴The individual cells were seeded in 96-well plates and placed in an incubator for 24h to adhere. Setting the concentration gradient of costunolide at 2 μ M, 5 μ M, 10 μ M, 20 μ M, 40 μ M, 60 μ M, and 80 μ M, adding corresponding concentration medicine according to groups, and culturing in incubator for 24 h. After the culture is finished, 20 mu L of MTT (5 mg. mL < -1 >) is added into each hole, the culture medium is sucked out after the light-shielding culture is carried out for 2h, 100 mu L of LDMSO is added into each hole, the absorbance is detected by using an enzyme labeling instrument after the full oscillation, the wavelength is 570nm, the result is recorded, and the bMSCs survival curve is drawn by taking the medicine concentration as the abscissa and the cell survival rate as the ordinate.

1.2.3 Costunolide induced bMSCs differentiation into neural-like cells

Select generation 3 bMSCs at 1.0 × 10 per well⁵The cells were seeded on 6-well plates and cultured in an incubator for 24 hours to adhere to the walls. According to MTT results, costunolide solution with the concentration of 10 mu M is selected to induce bMSCs, 3 groups of 4h, 12h and 24h are set according to time, the morphological change of cells is observed at regular time, and the cells are subjected to skeleton staining. And (3) fixing the cells after induction, sealing the cells, and detecting the expression quantity change of three nerve cell marker proteins including NSE, GFAP and Nestin by an immunofluorescence staining method.

1.2.4 preparation of animal model for cerebral ischemia-reperfusion

The 40 rats were randomly and equally divided into Sham surgery group (Sham), Model group (Model), MSC group and costunolide-induced MSC group (induced group)) (n-10). Production of Central cerebral artery occlusion (MCAO) focal cerebral ischemia model [12 ] Using modified ZeaLonga's Thrombus method]. The method comprises the following specific steps: the volume fraction of the chloral hydrate solution is 10 percent, and the concentration is 0.33-0.36 mL-100 g^-1After the abdominal cavity injection anesthesia is performed, the rat is fixed on an operation board, the neck is disinfected conventionally and then is preserved, a cut is made in the center of the neck, muscles are separated obtusely by a cotton swab, the right common carotid artery is exposed, the common carotid artery, the internal carotid artery and the external carotid artery are separated, the proximal end of the common carotid artery is ligated, the upper end of the common carotid artery is cut, a suture plug is inserted into the cut and is led in (the proximal end of the suture plug is marked at 1.5cm, and the marking point reaches the Y-shaped branch port during the line inlet), the suture and the disinfection are performed, 5mL of physiological saline is supplemented after each rat is operated, and the suture plug is pulled out after 2h for reperfusion.

The nerve function impairment scoring was performed according to the 5-point method of LongaZ, 1-2 animals were randomly assigned, and 2h after the line drawing, 1mL of bMSCs cells containing 1.0 × 106 cells or induced bMSCs cell suspension (PBS as solvent) was injected from the tail vein for each animal, and the sham-operated group and the model group were injected with the same amount of PBS. Animals were scored for neurological function at 2h, 1d, 3d, 5d, 7d post-surgery. After 7 days of molding, the rats were sacrificed and the brains were removed, subjected to TTC staining and HE staining, and the water content of the brain tissue was measured by dry-wet weight method.

1.2.5 statistical analysis

SPSS17.0 is adopted to carry out one-factor variance analysis on the data, and the data results are expressed as mean +/-standard deviation

Is represented by P<0.05 indicates significant difference and statistical significance.

2. Results

2.1 Costunolide toxicity assay for bMSCs

The survival rate of the bMSCs is detected by MTT experiment, the result is shown in figure 1, the survival rate of the cells is higher than 90% in the concentration range of 2-10 muM as the concentration of costunolide is increased, and the survival rate of the cells is obviously reduced when the concentration exceeds 10 muM. Shows that when the costunolide concentration is more than 10 mu M, the costunolide has larger cytotoxicity. Therefore, costunolide was chosen at a concentration of 10 μ M for the induction of bMSCs in this experiment.

2.2 morphological study of bMSCs after induction, wherein the morphological change of the bMSCs is used for evaluating the induction effect of costunolide on the bMSCs to neural-like cells. The observation result under the light microscope is shown in figure 2, and the cell shape is gradually elongated and cell branches appear from figure A to figure D within 0-24h, and the cell shows the nerve cell-like shape.

Further observation of cell morphology by cytoskeletal (. beta. -actin) staining revealed that the cells gradually changed from spindle-shaped, unbranched forms in FIG. A to longer, branched forms in FIG. D under the action of costunolide, and appeared as nerve cells, as shown in FIG. 3. The skeleton staining result is consistent with the observation result under the light microscope. Indicating that the bMSCs gradually differentiate towards neural-like cells under the condition induction of costunolide.

2.3 immunofluorescence staining for neural cell marker protein

The neuron-specific proteins NSE, Nestin and the astrocyte-specific protein GFAP are used as indexes for detecting whether the neural-like cells exist after the stem cells are induced to differentiate. The staining results are shown in fig. 4, compared with the blank group, the expression levels of three nerve cell marker proteins after costunolide induction of bMSCs are increased, wherein the expression levels of the proteins GFAP and Nestin reach peak values 24h after induction, and the expression level of the protein NSE reaches the maximum value 12h after induction. It was demonstrated that bMSCs gradually differentiated towards neural-like cells after induction with costunolide.

2.4 neurological impairment score

The postoperative animals were scored for neurological impairment according to LongaZ's 5-point score, and the results are shown in table 1, with the MSC group and induced (indeded MSC) animals scored somewhat lower within 2h and 1d after surgery, but with no significant difference (P > 0.05) compared to the model group; within 5d and 7d after operation, the MSC group score is obviously reduced compared with the model group (P <0.05), and the induced group score is more obviously different compared with the model group (P < 0.01).

TABLE 1 post-operative animal neurological impairment score

n＝10

Note: p <0.05, P < 0.01: comparing with the grading data of the neural function impairment of the corresponding model group on the same day)

2.5TTC staining, cerebral infarct volume and brain tissue Water content

After 7d, the cerebral infarction is shown in fig. 5, the cerebral infarction volume of rats in the MSC group and the induction group is obviously smaller than that of the model group, and the cerebral infarction volume of rats in the induction group is obviously reduced compared with that of the MSC group. Table 2 shows that the cerebral infarction volume of rats in the induced group is 14.76% ± 0.56, which is significantly reduced and significantly different (P <0.01) compared with the model group; the brain infarction volume of the MSC group is 23.15% + -0.08, and is reduced compared with the model group, but no significant difference exists (P is more than 0.05); the cerebral infarction volume of the rats in the induction group is also significantly reduced compared with the MSC group (P < 0.01). The water content of the rat brain tissue of the model group is 81.19% + -0.27, and the rat brain tissue is obviously increased and has significant difference (P <0.05) compared with a sham operation group; the water content of the brain tissue of the MSC group is 80.80% + -0.20, and the brain tissue is reduced compared with the model group, but has no significant difference (P is more than 0.05); the water content of the brain tissue of the rats in the induction group is 78.93% + -0.38, the brain tissue is obviously reduced compared with the model group, the significant difference is generated (P is less than 0.05), the significant difference is also generated (P is less than 0.05) compared with the MSC group, the bMSCs and the induced bMSCs can reduce the cerebral infarction volume of the rats with cerebral ischemia injury and reduce the water content of the brain, and the effect of the induction group is better than that of the MSC group.

TABLE 2 post-operative animal cerebral infarction volume and brain tissue water content variation

n＝10)

Note: p <0.05, P < 0.01; comparing to a model set; & P & lt, 0.05, compared to sham group; # P <0.05, # P <0.01, compared to MSC group

2.6 HE staining

The histological observation of the rat cerebral ischemia semi-dark area in fig. 6 shows that the neuron cell structure of the sham operation group is normal, the arrangement is neat and compact, the staining is uniform, and the cell nucleus is large and round; the model group has damaged neuron cell structure, and obvious solid shrinkage and deep staining of neuron cell nucleuses, interstitial edema and loose structure can be seen; both the MSC group and the induction group showed that the rat ischemic penumbra area was constricted, the number of densely infected neurons was reduced, and the interstitial edema was reduced, but the recovery effect of the ischemic penumbra area was superior to that of the MSC group in the two groups. The experimental results show that the induced bMSCs have more obvious treatment effect on cerebral ischemia-reperfusion injury.

Overall, Glial Fibrillary Acidic Protein (GFAP) is a marker of astrocyte activation; neuron-specific enolase (NSE) is an acid protease specific to neurons and neuroendocrine cells; nestin (Nestin) is an intermediate filament type of protein that is a characteristic marker of neural stem cells [23-24 ]. These three proteins are major marker proteins for demonstrating differentiation of stem cells into neural cells upon induction [14 ]. In the research, the fact that the cell morphology is gradually elongated and branched along with the prolonging of the induction time of costunolide on the bMSCs is found, the nerve-like cell morphology is shown, and meanwhile, the expression levels of three nerve cell marker proteins including NSE, GFAP and Nestin are also up-regulated, and the costunolide is proved to be capable of inducing the bMSCs to be differentiated to the nerve-like cells. On the basis, the treatment effect of the induced bMSCs is verified by utilizing a cerebral ischemia reperfusion rat model, and the nerve function damage score, the cerebral infarction volume, the brain water content and the brain tissue HE staining result show that the induction group and the MSC group can relieve the postoperative brain damage, and the treatment effect of the induction group is obviously superior to that of the MSC group by comparing the two treatment results.

The bMSCs can be differentiated to nerve-like cells after being induced by costunolide, and the damage of focus parts can be obviously reduced by treating cerebral ischemia by using the induced bMSCs. The stem cell therapy field still has unlimited potential, and better direction and thought can be provided for treating cardiovascular and cerebrovascular diseases in future, so that the stem cell therapy method has high clinical value.

Although the present invention has been described herein with reference to the illustrated embodiments thereof, the above-described embodiments are only one of the preferred embodiments of the present invention, and the embodiments of the present invention are not limited thereto, and it should be understood that many other modifications and embodiments can be devised by those skilled in the art that will fall within the spirit and scope of the principles of this disclosure.

Claims

1. The application of the compound shown as the formula (I) in inducing stem cells to differentiate into nerve-like cells by using the compound as an inducer, wherein the structural formula of the compound shown as the formula (I) is shown as follows

；

The stem cell is a bone marrow mesenchymal stem cell; the concentration of the inducer is 2-10 mu M.

2. Use according to claim 1, characterized in that: the inducer is used for up-regulating NSE, GFAP and Nestin nerve cell protein expression.

3. Use according to claim 1, characterized in that: the method for inducing the stem cells to differentiate into the neural-like cells comprises the following steps: and (3) taking the purified stem cells, and then placing the stem cells into a culture solution containing an inducer to culture for more than 12 hours to obtain the neural-like cells.

4. Use according to claim 3, characterized in that: the culture time is 12-24 h.

5. A method for inducing the differentiation of bone marrow mesenchymal stem cells into neural-like cells by using a compound as an inducer, which comprises the following steps: taking purified stem cells, and culturing in culture solution containing inducer for more than 12h to obtain the neural-like cells, wherein the compound formula of formula (I) is as follows

。