CN116870007B - Application of compound in preparation of medicine for treating diseases related to nerve axon injury - Google Patents
Application of compound in preparation of medicine for treating diseases related to nerve axon injuryInfo
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- CN116870007B CN116870007B CN202310886397.8A CN202310886397A CN116870007B CN 116870007 B CN116870007 B CN 116870007B CN 202310886397 A CN202310886397 A CN 202310886397A CN 116870007 B CN116870007 B CN 116870007B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
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Abstract
The invention discloses application of a compound in preparing a medicament for treating diseases related to nerve axon injury, wherein the chemical formula of the compound is shown as a formula (I). According to the invention, the research proves that the compound molecule Z52453295 can obviously promote the axon regeneration of dorsal root ganglion neurons and can obviously improve the axon regeneration after sciatic nerve injury, so that the compound molecule Z52453295 can be used as an important compound molecule for promoting the axon regeneration and repair after nerve injury, and has good curative effect in preparing medicines for promoting the axon regeneration after nerve injury.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to application of a compound in preparation of a medicament for treating diseases related to nerve axon injury.
Background
Nerve damage, particularly loss of rupture or long-distance defect of axons, is a common disease in modern life, causing serious physiological dysfunction and huge social and family burden to patients. The types of nerve injury mainly comprise peripheral nerve injury and central nerve injury, and the current strategy for treating the nerve injury is limited to the application of relying on the traditional autologous nerve transplantation operation and tissue engineering materials, but the treatment effect is poor and a plurality of problems exist, such as poor function recovery, autologous nerve source limitation and the like. Accordingly, research is being pursued to develop and screen for compounds for promoting regeneration after nerve axonal injury.
The regulation and control of the endogenous axon regeneration of neurons are the key to the regeneration and repair of the axon after nerve injury, and the current strategy for promoting the axon regeneration is mainly focused on the regulation and control of the expression of genes related to the axon regeneration, and the direct intervention of the expression of the genes has a plurality of risks, such as cancerogenic action and the like. Therefore, the preparation of the medicine for promoting the regeneration of the axons after the nerve injury by using the compound molecules has better potential advantages.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides an application of a compound in preparing a medicament for treating diseases related to nerve axon injury, and solves the problem of lack of compound molecules for promoting axon regeneration after nerve injury at present.
The invention is realized by the following technical scheme:
Use of a compound of formula (I), a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a disease associated with axonal injury, the compound having the formula:
preferably, the disease associated with neurite damage includes peripheral nerve damage and central nerve damage.
A pharmaceutical composition for treating diseases related to nerve axon injury comprises the compound, pharmaceutically acceptable salts thereof and pharmaceutically acceptable auxiliary materials thereof.
A pharmaceutical composition for promoting the repair of nerve axon injury comprises the compound, pharmaceutically acceptable salt thereof and pharmaceutically acceptable auxiliary materials thereof.
Preferably, the auxiliary materials comprise one or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers and lubricants.
Preferably, the pharmaceutical composition is in an oral or non-oral dosage form.
Preferably, the pharmaceutical composition is a tablet, capsule, powder, pill, granule, solution, suspension, syrup, injection, suppository, inhalant or spray.
The beneficial effects of the invention are as follows:
According to the invention, the toxicity of the compound molecule Z52453295 to dorsal root ganglion neurons is researched, the effect that the activity of rat dorsal root ganglion neurons can be obviously inhibited when the concentration of Z52453295 is 40-80 mu M is researched, and further analysis on the function of axon regeneration after neuron injury shows that the axon regeneration of dorsal root ganglion neurons can be obviously promoted by the concentration of Z52453295 of 10 mu M, and the axon regeneration after sciatic nerve injury can be obviously improved by the intrathecal injection of Z52453295 with the concentration of 6.25mM and 25mM, so that the compound molecule Z52453295 can be used as an important molecule for promoting axon regeneration repair after nerve injury and has good curative effect in preparing a medicine for promoting axon regeneration after nerve injury.
Drawings
FIG. 1 is a schematic diagram showing cytotoxicity of compound molecule Z52453295 at different concentrations on dorsal root ganglion neurons in example 2, wherein DMSO is used as a control;
FIG. 2 is a schematic diagram showing the effect of different concentrations of compound molecule Z52453295 on the regeneration of dorsal root ganglion neurites in example 3, wherein A is the regeneration of compound molecule Z52453295 on dorsal root ganglion neurites, B is a statistical plot of the number of neuronal neurites, C is a statistical plot of the total length of neuronal neurites, D is a statistical plot of the longest neuronal neurites, wherein DMSO is a control;
FIG. 3 is a schematic diagram showing the effect of different concentrations of compound molecule Z52453295 on axon regeneration after sciatic nerve injury in example 4, wherein A is the regeneration condition of compound molecule Z52453295 on sciatic nerve axon, B is a statistical graph of the regeneration length of axon after sciatic nerve injury, and DMSO is a control.
Detailed Description
The invention will be described in further detail with reference to the drawings and the specific embodiments.
Unless otherwise indicated, all technical means used in the following examples are conventional means well known to those skilled in the art, and experimental methods without specifying specific conditions are conventional in the art.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The quantitative experiments referred to in the following examples were each set up for at least three replicates and the results averaged.
The animals used in the following examples were SD rats, from the university of south-pass laboratory animal center.
Example 1
A compound molecule Z52453295 with the function of repairing nerve axon injury is named as Z52453295 (SC), CAS is 956935-02-7, the molecular formula is C 28H30N4 O, and the molecular weight is 438.564. Purchased from Enamine company.
The chemical formula of the compound molecule Z52453295 is shown in the following formula (I):
EXAMPLE 2 cytotoxic Effect of Compound molecule Z52453295 on dorsal root ganglion neurons
1. Rat dorsal root ganglion neuron separation culture
(1) The pre-chilled dissection solution was placed in a small dish, and placed on ice after the antibiotic was added. Abfacine (0.2 mL/10 g) was intraperitoneally injected to anesthetize the rats, the entire rat spinal column was isolated by aseptic technique, and after opening the lamina, all dorsal root ganglion tissues were removed using microshutter and placed in the anatomic solution. After rinsing the tissue 2-3 times with sterile PBS, 2mL of preheated collagenase (3 mg/mL) was added to the dorsal root ganglion tissue, and after the tissue was sufficiently sheared with micro-shears, it was digested for 90min in a cell incubator.
(2) The collagenase in the mixed solution is discarded by using a centrifugal method, then 1mL of preheated pancreatin (0.25%) digestive juice is added, and the mixture is blown and dispersed uniformly and then is put into a cell incubator to be digested continuously for 10min, and the blowing and the dispersing are repeated repeatedly and uniformly during the period. After the tissue was digested to no obvious tissue mass, 3mL of digestion stop solution was added to the centrifuge tube to terminate digestion.
(3) After passing through the screen (70 μm) the cell suspension was centrifuged at 1200rpm for 5min in a new centrifuge tube, and the supernatant was discarded. 4mL of pre-warmed 10% BSA solution was added to the centrifuge tube, the cells were resuspended and centrifuged at 900rpm for 5min. Repeating the above steps. Adding pre-heated neuron culture medium, blowing cells uniformly, inoculating into a cell culture plate pre-coated with Polylysine (PLL), cross mixing, and culturing in a culture box with 5% CO 2 and 37 ℃.
2. Detection of cytotoxicity of compound molecule Z52453295 on dorsal root ganglion neurons
The isolated dorsal root ganglion neurons were inoculated into 96-well cell culture plates (1 million cells per well), divided into 5 groups, each group was set to 4 multiplex wells, compound molecules Z52453295 with concentrations of 0, 5, 10, 20, 40, 80. Mu.M and control DMSO were added respectively, and after shaking uniformly cross-wise, they were placed in an incubator for 24 hours, CCK-8 detection reagent (10. Mu.L of CCK-8 reagent was added per 100. Mu.L of medium) was added, after mixing uniformly, they were placed in the incubator for continuous culture for 1 hour, and absorbance values were detected at a wavelength of 450 nm.
3. Experimental results
As shown in FIG. 1, in vitro, in the concentration range of 0-20 mu M, the compound molecule Z52453295 has no cytotoxicity on dorsal root ganglion neurons, and can remarkably inhibit the activity of neuron cells when the concentration reaches 40-80 mu M (** P < 0.01).
EXAMPLE 3 Effect of Compound molecule Z52453295 on dorsal root ganglion neuronal axon regeneration
1. Rat dorsal root ganglion neuron separation culture
The procedure is as in example 2.
2. Dorsal root ganglion neuron in vitro axon injury regeneration experiment
(1) After the dorsal root ganglion neurons are cultured in vitro for 48 hours, adding a precooled sterile PBS solution to rinse the cells for 2-3 times, adding preheated 0.025% pancreatin to digest for 5-10 minutes, adding 10% FBS to stop digestion, repeatedly blowing the cells, collecting cell suspension into a new 15mL centrifuge tube, and discarding the supernatant after the rotation speed is 1200rpm for 5 minutes.
(2) Adding a preheated neuron culture medium, blowing cells uniformly, inoculating the cells into a cell culture plate pre-coated with polylysine, simultaneously adding compound molecules Z52453295 with the concentration of 0, 10 and 20 mu M and contrast DMSO, mixing the cells uniformly in a cross manner, and then placing the cells into a 5% CO 2 and 37 ℃ incubator for culturing for 16-18 hours.
3. External immunofluorescence detection of dorsal root ganglion nerve cloud axon
(1) After pre-cooling the PBS to rinse the cells, pre-cooled 4% paraformaldehyde was added and the cells were fixed on ice for 20min.
(2) After formaldehyde was discarded, the solution was washed 5min×3 times with PBS at room temperature.
(3) Adding a sealing liquid, 200 mu L of sealing liquid in each hole, and standing at room temperature for 40min. The blocking solution was discarded, anti-Tuj1 antibody (1:1000) was diluted with primary Anti-dilution and incubated at 200. Mu.L per well at 4℃overnight.
(4) The primary antibody was discarded, rinsed with PBS, and washed 5min 3 times at room temperature. PBS was discarded, alexa fluor 647 coat anti-rabit (1:1000) diluted with secondary antibody diluent was gently added dropwise, 200. Mu.L per well was incubated at room temperature for about 2h in the absence of light. The secondary antibody was discarded and rinsed 5min x 3 times with PBS at room temperature. After the cleaning is finished, the round slide is picked out of the hole, one surface with cells is downwards covered on the slide glass with the sealing liquid, and the slide glass is placed in a wet box and stored at 4 ℃. Note that no bubbles are generated during the process. This procedure is protected from light. The number and length of axons regenerated by neurons were analyzed by photographing statistics.
4. Experimental results
As shown in FIG. 2, FIG. 2A shows the neurite outgrowth of neurons by different concentrations of compound molecule Z52453295, wherein DMSO is used as a control, and FIGS. 2B-D show statistics of the number, total length and longest neurites of neurons, respectively. As can be seen from fig. 2, the compound molecules Z52453295 at different concentrations have no significant effect on the number of axons of neurons, but can all promote regeneration of the axons of neurons to some extent, wherein the effect of the concentration of 10 μm is most significant, and the compound molecules Z52453295 at a concentration of 10 μm can significantly promote the total length (* P < 0.05) and the longest axon length (** P < 0.01) compared to the control group (DMSO).
EXAMPLE 4 Effect of Compound molecule Z52453295 on axonal regeneration following sciatic nerve injury
1. Establishing a rat sciatic nerve clamp injury model
Male C57BL/6J rats of about 8 weeks were selected for the experiment, 25 animals per batch were randomly divided into 5 groups, and supplied by the university laboratory animal center of Nantong. After weighing the grouped rats, the rats were anesthetized with avermectin, the bilateral sciatic nerves of the rats were aseptically isolated after preparing the hairs, and the wound surface was sutured after sciatic nerve clamping according to (54N/15 s) using a clamping forceps.
2. Intrathecal injection of Compound molecule Z52453295
(1) After the rats were subjected to sciatic nerve clamping injury, intrathecal injection of the compound molecules was performed. After the skin of the lumbar spine of the rat is cut off and the spinal spinous process of the horsetail of the rat is cut off, 4-5 mu L of compound molecule Z52453295 is sucked by a microinjector to intrathecal injection, and the concentration is respectively 0, 1.56, 6.25, 25 and 100mM in the injection process.
(2) The tail flick action of the rat is seen to be successful after the glass electrode is penetrated into the spinal space, the injection is carried out by using a microsyringe according to the speed of 50nL/s, the needle head is stopped for about 30s after the injection is finished, so that liquid leakage is prevented, and then the needle head is slowly pulled out to suture the wound surface. After the end, the rats are placed on an electric blanket, and after the rats wake up, the rats are put back into the cage box.
3. Staining marker for sciatic nerve regeneration axon
On the third day after step 2 was completed, rats were anesthetized and then perfused with 4% paraformaldehyde solution for sampling and fixed overnight at room temperature. The sciatic nerve was dehydrated and embedded at 30%, and frozen sections (16 μm) were prepared. Immunofluorescence assay first, slice samples were washed 5min x 3 times at 0.3% TBST room temperature, blocked 1h at 5% BSA room temperature, incubated for one-antigen SCG10 (1:1000) overnight at 4 ℃, incubated for 2h at room temperature protected from light for fluorescent secondary antibody Mouse-594 (1:1000), photographed under fluorescent microscope, and statistically analyzed for length of regenerated sciatic nerve axons. SCG10 is a marker protein for regeneration of nerve axons.
4. Experimental results
As shown in fig. 3, fig. 3 a is a plot of axonal regeneration after intrathecal injection of different concentrations of compound molecule Z52453295 against sciatic nerve injury, wherein DMSO is a control and fig. 3B is a statistical plot of sciatic nerve longest regenerated axons. The results show that compound molecule Z52453295 with intrathecal injection concentration of 6.25mM (** P < 0.01) or 25mM (* P < 0.05) can significantly promote regeneration of axons after sciatic nerve injury, while compound molecule Z52453295 with concentration of 1.56mM and 100mM has no significant effect on regeneration of sciatic nerve axons.
In conclusion, the application of the compound molecule Z52453295 in preparing the medicine for treating the diseases related to the nerve axon injury is provided, and experiments in vitro and in vivo show that the compound molecule Z52453295 has no cytotoxicity on dorsal root ganglion neurons in a concentration range of 0-20 mu M, and can obviously inhibit the activity of neuron cells when the concentration reaches 40-80 mu M. 10 mu M of compound molecule Z52453295 can significantly promote the regeneration of sciatic nerve dorsal root ganglion neuron axons in peripheral nerves, and 6.25mM or 25mM of compound molecule Z52453295 can significantly promote the regeneration of axons after sciatic nerve injury. Thus, compound molecule Z52453295 can be an important compound molecule that promotes regeneration and repair of axons following nerve injury.
The above embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by a person skilled in the art without any inventive effort, are intended to be within the scope of the present invention based on the embodiments of the present invention.
Claims (1)
1. The application of a compound shown in a formula (I) and pharmaceutically acceptable salts thereof in preparing a medicament for treating peripheral nerve injury is characterized in that the chemical formula of the compound is as follows:
formula (I).
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