阅读说明：本技术 去氢骆驼蓬碱在促进神经再生和修复神经损伤方面的应用 (Application of harmine in promoting nerve regeneration and repairing nerve injury ) 是由 周炳 柳瑞璇 杨晓燕 于 2021-10-15 设计创作，主要内容包括：本发明公开了去氢骆驼蓬碱在制备促进神经再生和修复神经损伤药物方面的应用。本发明利用微流控系统在体外培养皮层神经元和DRG神经元,形成中枢神经系统和周围神经系统的神经轴突损伤后,采用去氢骆驼蓬碱进行治疗,发现去氢骆驼蓬碱可以显著促进皮层神经元和DRG神经元轴突的损伤后的再生,该发现为脊髓损伤和外周损伤等神经损伤患者的治疗提供了新的方向。(The invention discloses application of harmine in preparing a medicament for promoting nerve regeneration and repairing nerve injury. In the invention, the cortical neuron and the DRG neuron are cultured in vitro by utilizing the microfluidic system to form the nerve axon injury of the central nervous system and the peripheral nervous system, and then the harms are treated by using the harmine, so that the harmine can remarkably promote the regeneration of the damaged cortical neuron and the DRG neuron axon after discovery, and the discovery provides a new direction for the treatment of nerve injury patients such as spinal cord injury, peripheral injury and the like.)

1. Application of harmine in preparing medicine for promoting nerve regeneration and repairing nerve injury is provided.

2. The use of harmine according to claim 1 for the manufacture of a medicament for promoting nerve regeneration and repairing nerve injury, wherein nerve regeneration and nerve injury are neuronal axon regeneration and neuronal axon injury.

3. The use of harmine according to claim 2, wherein the neuronal axonal damage is central neuronal axonal damage in the manufacture of a medicament for promoting nerve regeneration and repairing nerve damage.

4. The use of harmine according to claim 2, wherein the neuronal axonal damage is peripheral neuronal axonal damage in the manufacture of a medicament for promoting nerve regeneration and repairing nerve damage.

5. The use of Harmine in the manufacture of a medicament for promoting nerve regeneration and repairing nerve damage according to claim 1, wherein Harmine (Harmine), CAS No.442-51-3, has a structural formula shown in formula I:

6. application of salt, ester or derivative of harmine in preparing medicine for promoting nerve regeneration and repairing nerve injury is provided.

7. The use of a salt, ester or derivative of harmine according to claim 6, wherein the nerve regeneration and nerve injury is neuronal axon regeneration and neuronal axon injury.

8. The use of a salt, ester or derivative of harmine according to claim 7 for the manufacture of a medicament for promoting nerve regeneration and repairing nerve damage, wherein the neuronal axonal damage is a central neuronal axonal damage or a peripheral neuronal axonal damage.

9. The use of a salt, ester or derivative of Harmine according to claim 6 in the manufacture of a medicament for promoting nerve regeneration and repairing nerve damage, wherein Harmine (Harmine), CAS No.442-51-3, has the formula I:

Technical Field

The invention belongs to the field of medicines, relates to application of harmine in promoting nerve regeneration and repairing nerve injury, and particularly relates to application of harmine in preparing a medicine for promoting nerve regeneration and repairing nerve injury.

Background

Nerve injuries mainly include spinal cord injuries and peripheral nerve injuries, and particularly spinal cord injuries can cause irreversible injuries. The axon of the neuron usually has a super-long structure, and nerve injury causes interruption of nerve signals at the damaged part, influences the normal function of the nervous system and has high disability rate. Neurons are highly differentiated cells that do not have the ability to divide and regenerate themselves, and therefore the field of regeneration of injuries to the nervous system has been devoted to research on the regeneration of neuronal axons.

The axons of neurons of the peripheral nervous system have a certain ability to regenerate after injury, and the lost function can be partially recovered. However, adult central nervous system neurons do not have axonal regeneration capacity, and severe injury often results in permanent functional deficits. Therefore, how to regenerate nerve injury is a main problem which needs to be solved urgently at present, but the clinical realization of axon regeneration and functional recovery at present is not ideal. Therefore, the development of a drug which can promote axon regeneration after nerve injury is urgently needed, and the drug has great significance for the treatment of nerve injury.

Disclosure of Invention

Aiming at the defects in the prior art, the application of harmine or pharmaceutically acceptable salts, esters and derivatives thereof in promoting nerve regeneration and repairing nerve injury is provided, and the specific technical scheme is as follows:

the embodiment of the invention provides application of harmine in preparing a medicament for promoting nerve regeneration and repairing nerve injury.

The embodiment of the invention also provides application of the salt, ester or derivative of harmine in preparing medicines for promoting nerve regeneration and repairing nerve injury.

Preferably, the nerve regeneration and nerve injury is neuronal axon regeneration and neuronal axon injury.

Preferably, the neuronal axonal injury is an axonal injury of a central neuron.

Preferably, the neuronal axonal injury is a peripheral neuronal axonal injury.

Preferably, the Harmine (Harmine), CAS No.442-51-3, has a structural formula shown in formula I:

compared with the prior art, the invention has the following beneficial effects:

the invention originally discovers that harmine can promote the protrusion growth of central neurons and the axon regrowth after the central neurons and peripheral neurons are damaged. Therefore, the medicine prepared by the harmine can participate in the repair of peripheral nerve injury by promoting the axon regrowth of DRG neurons and participate in the repair of central nerve injury by promoting the axon regrowth of cortical neurons, and provides a new target point for the treatment of nerve injury diseases.

Drawings

The invention may be better understood by reference to the following drawings. The components in the figures are not to be considered as drawn to scale, emphasis instead being placed upon illustrating the principles of the invention.

FIG. 1 is a schematic diagram of axon regeneration after neuron culture and injury in the microfluidic system used in example 1 of the present invention;

FIG. 2 is a graph showing that harmine promotes the outgrowth of cortical neurons in example 1 of the present invention;

FIG. 3 is a graph of harmine-treated cortical neuron axon regeneration after injury in example 2 of the present invention;

FIG. 4 shows that harmine promotes axonal regeneration in DRG neurons after injury in example 3 of the invention.

Detailed Description

The present invention is further described in the following description with reference to the drawings and the detailed embodiments so that those skilled in the art can better understand the present invention and can practice the present invention, but the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

The harmine used in the examples below was a DMSO solution of harmine.

1. In vitro culture method of cortical neurons

(1) Prior to cell inoculation, sterile 25mm diameter round slides were placed in 35mm petri dishes in a clean bench and 2ml of a mixture of poly-D-lysine hydrobromide (l 00. mu.g/ml, Sigma-Aldrich) and laminin (Roche) was added at 37 ℃ in 5% CO₂And coating in a cell culture box for 2 h.

(2) And (3) dissection: the brains of young SD rats less than 12h after birth were isolated, the meninges were stripped off under a microscope with curved forceps, the cortical neural tissue was removed from the brains with curved forceps, and the tissue was minced and placed in a 15ml centrifuge tube.

(3) Digestion: adding 5ml of papain (Worthington) dissolved in HBSS (Gibco) into the 15ml centrifuge tube, gently blowing and mixing the precipitated cells in the centrifuge tube; then, the tube was centrifuged at 37 ℃ and 5% CO₂Horizontally placing in an incubator for 40min, then blowing and beating cell sap for 10-15 times by using a 5ml pipette, and finally centrifuging in a centrifugal tube at the rotating speed of 300rpm for 5 min;

(4) preparing a neuron cell suspension: taking out the digestive juice, resuspending the cells with 5ml of 1:1 solution, standing the larger tissue block for 5min, then centrifuging the suspension by a centrifuge at the rotating speed of 300rpm for 10min, discarding the supernatant after centrifugation, and adding a neuron basal Medium ((Neurobasal-A Medium, B27 Supplement, GlutaMAX Supplement, fetal calf serum and penicillin-streptomycin) for resuspension to obtain primary neuron cell suspension;

(5) grafting cortical neurons: taking 10 μ l of the blown and uniformly mixed cortical neuron cell suspension for cell counting, inoculating the cell suspension into a microfluidic device according to the quantity of 3 ten thousand per culture dish, adding a cortical neuron basal medium, and placing the cell suspension at 37 ℃ and 5% CO₂The cells were cultured in a cell incubator for 7 days.

In vitro culture method of DRG neuron

(1) Prior to cell inoculation, sterile 25mm diameter round slides were placed in 35mm petri dishes in a clean bench and 2ml poly-D-lysine hydrobromide (l 00. mu.g/ml, Sigma-Aldrich) and laminin (Roche) were addedMixture solution, 5% CO at 37 ℃₂And coating in a cell culture box for 2 h.

(2) Material taking: after the SD rats of P8 were anesthetized, the entire spine was removed with scissors and the spinal cord was exposed by cutting from the midline. After the spinal cord was stripped, the DRG was removed with forceps and the central and peripheral axons were trimmed off.

(3) Digestion: adding into solution of dispase (Yeasen) and collagenase (Worthington), adding 5% CO at 37 deg.C₂The cell culture box was digested for 30 min.

(4) Preparing a neuron cell suspension: the digested DRG was gently and evenly blown, left for 10min to pellet large pieces of tissue, and then the cell suspension was transferred into 1.5ml and centrifuged at 300rpm for 3min by a centrifuge. After centrifugation, the supernatant was discarded, and the DRG neuronal basal Medium (Neurobasal Medium, B27 Supplement, GlutaMAX Supplement, and penicillin-streptomycin) was resuspended to obtain a DRG neuronal cell suspension.

(5) Inoculation of DRG neurons: taking 10 mul of the uniformly blown DRG neuron cell suspension for cell counting, inoculating the cell suspension into a microfluidic device according to the number of 3 ten thousand per hole, adding a DRG neuron basal medium, and placing the cell suspension at 37 ℃ and 5% CO₂The cells were cultured in a cell incubator for 7 days.

3. Establishing cortical neuron damage model

The microfluidic device separates two sides of the cell body and the axon of the cortical neuron, and the axon of the cortical neuron cultured for 7 days grows into the contralateral chamber along the micro-channel of the microfluidic device. Cutting off axon by vacuum suction, adding harmine or basic culture medium, and culturing for 48 h.

4. Establishing DRG neuron damage model

The microfluidic device separates the cell body and the axon of the DRG neuron, and the axon of the DRG neuron cultured for 7 days grows into the contralateral chamber along the micro-channel of the microfluidic device. The axons were cut off by vacuum suction.

Example 1: harmine for promoting growth of cortical neuron protrusion

The cortical neuron cell suspension is cultured in a microfluidic system consisting of a microfluidic device and a round slide. The system is characterized in that a micro-flow channel is formed by photoetching treatment of an SU-8 silicon wafer master, and is prepared by pouring a PDMS material (SYLGARD 184 silicone adhesive and a catalyst are mixed according to a ratio of 10:1 (Thinky mixed liquid ARF-310 is treated and mixed and defoamed in two steps), Bel-Art vacuum defoaming and 80-degree drying for 2 hours, wherein the effect is shown in figure 1. when the system (Microfluidic chamber) is applied, a neuron cell body (somachamber) and an axon far end (axonal chamber) can be effectively separated, and after the axon far end (axonal chamber) is cut off by a vacuum pump, a regenerated neuron axon is dyed (beta III-Tubulin, red), can be well distinguished from the original neuron axon and can be quantitatively analyzed.

Experiment neurons were incubated at 37 ℃ and 5% CO₂The cells were cultured in a cell incubator for 2 days. After 2 days of in vitro culture, harmine was added to cortical neurons, and the culture continued for 48h after placement in the incubator. Cortical neurons were fixed with 4% PFA (Sigma-Aldrich), stained with Anti- β III Tubulin mAb (Promega, 1:500) primary and fluorescent secondary antibodies 488(1:1000) for neuron-specific immunofluorescence, and it was found that treatment of cortical neurons with dehydroharmine significantly enhanced the outgrowth of cortical neurons. At baseline levels, peganine-treated cortical neuronal projections increased 1.3-fold compared to controls (as shown in figure 2), while there was no significant difference in cell density after peganine-treated neurons. The results show that harmine can promote the growth of the processes of central neurons without impairing the survival of neurons.

Example 2: harmine-free method for promoting regeneration of axon of cortical neuron after injury

Culturing cortical neuron cell suspension in a microfluidic system composed of a microfluidic device and a round glass slide, and placing the microfluidic system at 37 ℃ and 5% CO₂Culturing in a cell culture box. Cutting off axon of cortical neuron cultured in vitro for 7 days by means of a vacuum pump, adding harmine, and continuously culturing for 48 h. Cortical neurons were fixed with 4% PFA (Sigma-Aldrich) and Anti- β III TubulinmAb (Promega, 1:500) primary and fluorescent secondary antibodies 488(Life Technologies, 1:1000) or secondary antibodies 555(1:1000) split the cellular side and axonal side, stained for neuron-specific immunofluorescence, and the injured cortical neurons were treated with harmine, and it was found that harmine significantly enhanced the axonal regeneration of cortical neurons. In regenerative conditions, there was a 3-fold increase in cortical neuronal axons following harmfulness of harmfulness from harmfuline treatment (as shown in figure 3) compared to controls. The results indicate that harmine can promote axonal regeneration of injured central neurons.

Example 3: harmine-free method for promoting regeneration of injured DRG neuron axons

Culturing DRG neuron cell suspension in a microfluidic system composed of a microfluidic device and a round glass slide, and placing at 37 deg.C and 5% CO₂Culturing in a cell culture box. Cutting off the DRG neuron axons cultured in vitro for 7 days by a vacuum pump, adding harmine, and continuously culturing for 48 h. DRG neurons were fixed with 4% PFA (Sigma-Aldrich), stained specifically immunofluorescently with Anti- β III Tubulin mAb (Promega, 1:500) primary and fluorescent secondary antibodies 488(Life Technologies, 1:1000) for neurons, and treated post-injury DRG neurons with Peganine, which was found to significantly enhance axonal regeneration of DRG neurons. DRG neuronal axons increased 1.35 fold after harmfulness of peganine treatment compared to the control group (as shown in figure 3). The results indicate that harmine can promote axonal regeneration of injured peripheral neurons.

It is to be understood that the foregoing is merely illustrative of some embodiments and that changes, modifications, additions and/or variations may be made without departing from the scope and spirit of the disclosed embodiments, which are intended to be illustrative and not limiting. Furthermore, the described embodiments are directed to embodiments presently contemplated to be the most practical and preferred, it being understood that the embodiments should not be limited to the disclosed embodiments, but on the contrary, are intended to cover various modifications and equivalent arrangements included within the spirit and scope of the embodiments. Moreover, the various embodiments described above can be used in conjunction with other embodiments, e.g., aspects of one embodiment can be combined with aspects of another embodiment to realize yet another embodiment. In addition, each individual feature or element of any given assembly may constitute additional embodiments.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.