Application of guanine and isoguanine in preparation of medicine for treating or repairing peripheral nerve injury

Abstract

The invention provides an application of guanine and isoguanine in preparation of a drug for treating or repairing peripheral nerve injury, relates to the technical field of biomedicine, and provides an application of guanine Guanine and / or isoguanine Isoguanine in preparation of a drug for treating or repairing peripheral nerve injury. The specific embodiment verifies that the growth of DRG neuron axons can be obviously promoted by adding guanine and isoguanine into DRG neuron resuspended in vitro, and guanine and isoguanine in the application can regulate the growth of the DRG neuron axons in the peripheral nerve injury and regeneration process; the important effects of Guanine and Isoguanine in the nerve injury repairing process can be better understood, and meanwhile, the effectiveness of the medicine for repairing or treating peripheral nerve injury is verified; and a new target spot is provided for treatment of related nervous system diseases.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to the application of guanine and isoguanine in the preparation of medicines for treating or repairing peripheral nerve damage. Background Art

[0002] Peripheral nerve injury refers to the damage of nerve trunks, nerve plexuses or nerve endings due to various reasons (such as trauma, traction, compression, etc.), resulting in abnormal nerve function. This injury may cause the patient's sensory function, muscle movement function and autonomic nerve function to be impaired.

[0003] Currently, the treatments for peripheral nerve injury mainly include surgery, medication and physical therapy. Surgery is often used in cases of nerve rupture or severe compression to restore nerve function by repairing or reconstructing damaged nerves. Medication may involve analgesics, anti-inflammatory drugs and neurotrophic drugs to relieve pain, reduce inflammation and promote nerve function recovery. Physical therapy includes massage, electrical stimulation, thermotherapy, etc., which aims to help restore muscle and nerve function.

[0004] The activation of the intrinsic regenerative capacity of neurons after peripheral nerve injury is affected by many factors, including the metabolites of the cell body and surrounding microenvironment after neuronal injury. Seeking key metabolites related to promoting axon regeneration in the peripheral nervous system will help regenerate and repair peripheral nerves after injury. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the prior art lacks key metabolites related to promoting the regeneration of axons of peripheral nervous system neurons.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of guanine and / or isoguanine in preparing medicine for treating or repairing peripheral nerve damage.

[0008] Preferably, the peripheral nerve injury comprises sciatic nerve injury.

[0009] Preferably, the drug treats or repairs peripheral nerve damage by promoting the growth of DRG neuron axons.

[0010] The present application also provides a drug for treating or repairing peripheral nerve damage, wherein the drug at least comprises guanine and / or isoguanine.

[0011] Preferably, the medicine also includes other medically acceptable auxiliary agents.

[0012] Compared with the prior art, this application has at least the following beneficial effects:

[0013] (1) The present invention proposes the use of guanine and isoguanine in the preparation of drugs for treating or repairing peripheral nerve injury. The addition of guanine or isoguanine promotes the growth of DRG neuron axons. It also proposes drugs for repairing or treating peripheral nerve injury, providing new therapeutic drugs for the treatment of peripheral nerve injury.

[0014] (2) The embodiments of the present invention propose that adding Guanine or Isoguanine to DRG neurons resuspended in vitro can significantly promote the growth of DRG neuron axons; Guanine and Isoguanine in the present invention can regulate the growth of DRG neuron axons during peripheral nerve injury and regeneration, which helps to better understand the important role of Guanine and Isoguanine in the process of nerve injury and repair, and at the same time verifies the effectiveness of drugs for repairing or treating peripheral nerve injury; and provides new targets for the treatment of related nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure is a diagram of the cell viability level of DRG neuron cells after culture in medium containing different concentrations of Guanine and Isoguanine detected by CCK8 method in Example 1 of the present invention. Different concentrations of Guanine and Isoguanine have no significant effect on the viability level of DRG neuron cells.

[0016] Figure 2 This is a diagram showing the growth of axons of DRG neurons cultured in vitro by a culture medium containing Guanine or Isoguanine in Example 1 of the present invention; wherein, Figure 2 A is the growth diagram of DRG neuron axons after pre-injury treated with different concentrations of Guanine and Isoguanine, and anti-Tuj1 marked the axons; Figure 2 B and 2C are statistical graphs showing the effects of Guanine and Isoguanine on axonal growth of DRG neurons under pre-injury conditions.

[0017] Figure 3 This is a diagram showing the effect of intraperitoneal injection of Guanine and Isoguanine on the growth of axons in rats after sciatic nerve injury as described in Example 2 of the present invention; wherein, Figure 3 A is the animal experiment flow chart; Figure 3 B is a graph showing the growth of axons after sciatic nerve injury in rats detected by immunohistochemistry after intraperitoneal injection of Guanine or Isoguanine. Anti-SCG10 marks the regenerated axons of DRG neurons. Figure 3 C is the statistical graph of fluorescence intensity at different distances from the injury point according to the immunohistochemical results; Figure 3 D is the statistical result of axon regeneration coefficient after intraperitoneal injection of Guanine or Isoguanine. DETAILED DESCRIPTION

[0018] The present invention is further described in detail below in conjunction with specific embodiments.

[0019] The present application provides the use of guanine and / or isoguanine in the preparation of a drug for treating or repairing peripheral nerve damage. In one embodiment, the peripheral nerve damage includes sciatic nerve damage. The drug treats or repairs peripheral nerve damage by promoting the growth of DRG neuron axons.

[0020] Based on this, the present application also provides a drug for treating or repairing peripheral nerve damage, wherein the drug at least includes guanine and / or isoguanine. In one embodiment, the drug also includes other medically acceptable auxiliary agents.

[0021] The above contents are verified and explained in combination with specific verification experiments below.

[0022] Embodiment 1: Guanine and Isoguanine treatment promotes axonal growth of DRG neurons in vitro

[0023] 1-1. Extraction of primary DRG neurons

[0024] Several adult SD rats were anesthetized with a compound anesthetic, the back hair was shaved, and 75% alcohol was used for disinfection. The rats were dissected from the back and the complete spine was separated and removed. The upper vertebral plate was opened, and all the DRGs were removed and placed in a dish with Hibernate A (HA+1% PS) added. After all the DRGs were removed, the HA was discarded, and the rats were washed twice with PBS. The PBS was discarded, and a small amount of collagenase was added. The DRGs were fully chopped with ophthalmic scissors, and collagenase was added to an appropriate amount (1 mL for every DRG of 3 rats), and the rats were placed in a 37°C, 5% CO2 constant temperature cell culture incubator for digestion for 90 minutes. All the liquid in the dish was transferred to 5 mL EP tube, centrifuge at 1200rpm×5min, discard the supernatant, add 0.25% Trypsin-EDTA (pancreatin) preheated at 37℃ with the same amount of collagenase, blow 10-20 times with a 1mL pipette, put it back to 37℃, 5% CO2 constant temperature cell culture incubator for 3min, take it out and continue to blow 10-20 times, and the DRG tissue block can be completely digested; add 3 times the volume of trypsin digestion stop solution (10% FBS in PBS), blow and mix with a 1mL pipette; filter all the liquid through a 70μm mesh and collect the cell filtrate; centrifuge the collected cell filtrate at 1200rpm×5min, discard the supernatant, add 10mL 15% BSA, blow evenly with a 1mL pipette, centrifuge at 900rpm×5min, discard the supernatant and floating and suspended substances, and then add 7mL 15% BSA, blow evenly, centrifuge at 900rpm×5min, discard the supernatant and floating and suspended substances; add appropriate amount of adult rat dorsal root ganglion neuron cell (DRG) culture medium (NB+) and blow evenly the purified cells, evenly inoculate them into PLL-coated 24-well plates, and finally culture them in a 37°C, 5% CO2 constant temperature cell culture incubator.

[0025] 1-2. CCK8 assay to detect the viability of DRG neurons treated with guanine and isoguanine

[0026] Primary DRG neuron cells extracted in 1-1 were seeded into 96-well plates, cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 for 24 hours, the original culture medium was discarded, 10pM, 100pM, 1nM, 10nM, 100nM or 1μM Guanine and Isoguanine and their controls (equal volumes of complete culture medium) were added to the cells, 200μl / well, and 3 replicates were set under each treatment condition. After 24 hours of culture, the culture medium was discarded, and a culture medium containing 10% CCK8 reagent was added, and the cells were cultured in a constant temperature cell culture incubator at 37°C and 5% CO2 for 2 hours to collect the cells, and the absorbance of the cells in each well at 450nm was detected by an enzyme marker, and the cell viability of each well was counted.

[0027] The CCK8 method was used to detect the cell viability of DRG neurons cultured in vitro after treatment with different concentrations of Guanine and Isoguanine. Figure 1 As shown, Guanine and Isoguanine treatment did not affect the cell viability of DRG neurons in vitro. Statistical test method: one-way ANOVA.

[0028] 1-3. In vitro injury model of primary DRG neurons

[0029] After culturing neurons in 1-1 for 60-72 hours, discard the original culture medium, add 1 mL of 0.025% trypsin pre-warmed at 37°C to each well, place the well plate in a cell culture incubator, and digest for 3-5 minutes. Add 10% FBS to terminate the digestion, 2 mL / well. Use a gun to blow repeatedly to terminate the digestion until the adherent cells are suspended and basically dispersed into single cells. Transfer the cell suspension in the well plate to a sterile 5 mL centrifuge tube and centrifuge at 1200 rpm × 5 min. Discard the supernatant, add an appropriate amount of complete neuronal culture medium to resuspend the cell pellet, and inoculate it in a 24-well plate coated in advance with a round glass slide, 400 μl / well, with about 5000-6000 cells per well.

[0030] 1-4. Guanine and Isoguanine treatment of injured DRG neurons

[0031] After the cells were resuspended, 10 nM or 1 μM Guanine or Isoguanine and their controls (equal volumes of dilution medium) were immediately added to the cells, 500 μl / well, and 3 replicate wells were set up under each treatment condition. The length of neuronal axon growth was observed, and the cells were harvested after 24 hours for cell immunofluorescence experiments.

[0032] 1-5. Cell Immunofluorescence

[0033] Discard the cell culture medium, gently add 0.5mL 1×PBS to wash away the culture medium, and let stand at room temperature for 5min; discard 1×PBS, add pre-warmed 4% paraformaldehyde, and fix at room temperature for 15min; discard 4% paraformaldehyde, add 0.5mL 1×PBS to wash away paraformaldehyde, let stand at room temperature, 5min / time, wash twice; discard 1×PBS, add 0.5mL 0.1% PBST to break the membrane, let stand at room temperature for 10min; discard 0.1% PBST, add immunohistochemistry blocking solution, 200μl / well, block at room temperature for 1h; discard blocking solution, dilute primary antibody with immunohistochemistry primary antibody diluent (Biyuntian), add primary antibody anti-Tuj1 (Abcam, ab18207, 1:500), and incubate overnight at 4℃;

[0034] Take the plate out of 4℃ and warm it up at room temperature for 0.5h; discard the primary antibody, add 1×PBS, let it stand at room temperature, wash 3 times for 10min / time; discard 1×PBS, protect from light, dilute the secondary antibody with immunohistochemistry secondary antibody diluent (Biyuntian), add secondary antibody Goat anti-RabbitIgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 594 (ThermoFisherScientific, A11012, 1:500), incubate at room temperature in the dark for 2 hours; discard the secondary antibody, add 1×PBS, stand at room temperature, wash 3 times for 10 minutes each time; in the dark, pick out the round glass slide with cells, seal it with DAPI-containing sealing solution (Biyuntian), place it in a dry dark box, observe it under a ZEISS upright fluorescence microscope, and take pictures. Observe the growth of the neurites, take pictures and save them, and count the longest neurite length and the distribution of neurite length in each group.

[0035] Different concentrations of Guanine and Isoguanine were used to treat pre-injured DRG neurons in vitro. Anti-Tuj1 antibody was used to label the neuronal cytoskeleton. The scale bar is 100 μm. Figure 2 As shown in A; the effects of Guanine and Isoguanine on the total length and maximum length of DRG neuron axons cultured in vitro under pre-injury conditions. Figure 2 B. Therefore, according to Figure 2 It can be seen that compared with the control group, Guanine and Isoguanine can significantly promote the regeneration of pre-injured DRG neuron axons in vitro.

[0036] Example 2: Guanine and Isoguanine treatment in vivo promotes the repair of sciatic nerve injury

[0037] Fifteen healthy adult male SD rats, weighing 200±20g, were randomly divided into three groups, with 5 rats in each group. The rats were anesthetized by intraperitoneal injection of compound anesthetic (0.35mL / 100g), the left hind limb was shaved and disinfected with iodine, the sciatic nerve in the middle of the left femur was surgically exposed, the nerve was separated from the muscle and basement membrane, and the middle part of the sciatic nerve was clamped with special forceps with full teeth. The clamping force was 54 Newtons and the time was 30s. After the clamping (including the day of the clamping), the rats were divided into three groups and given 0 / 10μM Guanine / 10μM Isoguanine (dissolved in saline) intraperitoneal injection (2mL / kg) every day, and the rats were killed on the fourth day (animal experimental process, as shown in the figure). Figure 3The injured sciatic nerve was removed and embedded in OCT and frozen. The primary antibody was anti-SCG10 (Novus, NBP1-49461, 1:400). The secondary antibody was Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, AlexaFluor TM 594 (ThermoFisher Scientific, A11012, 1:500), to label the regenerating axons of DRG neurons and to detect the growth of axons after sciatic nerve injury.

[0038] Immunohistochemical examination of axon regeneration after sciatic nerve injury in rats after intraperitoneal injection of Guanine and Isoguanine. Figure 3 As shown in B, anti-SCG10 marks the regenerated axons, and the scale bar is 500 μm; immunohistochemistry detects the relative fluorescence intensity of the tissue at different distances from the injury point. Figure 3 C. Statistical results of axon regeneration coefficient after intraperitoneal injection of Guanine and Isoguanine, as shown in Figure 3 Shown in D; Statistical test method: one-way ANOVA. Figure 3 B. Figure 3 In C and 3D, the results of axonal SCG10 immunohistochemistry showed that in vivo administration of Guanine and Isoguanine could significantly promote the regrowth of DRG neuron axons after sciatic nerve injury in rats.

[0039] Therefore, the results are Figure 3 As shown, guanine and isoguanine treatment can significantly promote the regeneration of DRG neuron axons after sciatic nerve crush injury.

[0040] The following are the experimental materials and their sources used in the examples of this application:

[0041]

[0042]

[0043] In summary, the application proposes the use of guanine and isoguanine in the preparation of drugs for treating or repairing peripheral nerve injury. Through specific examples, adding guanine and isoguanine to DRG neurons resuspended in vitro can significantly promote the growth of DRG neuron axons. Moreover, guanine and isoguanine in the application can regulate the axon growth of DRG neuron cells during peripheral nerve injury and regeneration, which helps to better understand the important role of Guanine and Isoguanine in the process of nerve injury and repair, and at the same time verifies the effectiveness of drugs for repairing or treating peripheral nerve injury; providing new targets for the treatment of related nervous system diseases.

Claims

1. Use of guanine and / or isoguanine in the preparation of drugs for treating or repairing peripheral nerve damage.

2. The use of guanine and / or isoguanine according to claim 1 in the preparation of a drug for treating or repairing peripheral nerve damage, characterized in that: The peripheral nerve injury includes sciatic nerve injury.

3. The use of guanine and / or isoguanine according to claim 1 in the preparation of a drug for treating or repairing peripheral nerve damage, characterized in that: The drug is used to treat or repair peripheral nerve damage by promoting the growth of DRG neuron axons.

4. A drug for treating or repairing peripheral nerve damage, characterized in that: The drug at least includes guanine and / or isoguanine.

5. A drug for treating or repairing peripheral nerve damage according to claim 4, characterized in that: The medicine also includes other medically acceptable auxiliary agents.

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