Use of guanine and isoguanine in the preparation of a medicament for treating or repairing peripheral nerve injury

Abstract

The application provides application of guanine and isoguanine in preparation of drugs for treating or repairing peripheral nerve injury, and relates to the technical field of biomedicine, and the technical scheme is characterized in that the application provides application of guanine Guanine and / or isoguanine Isoguanine in preparation of drugs for treating or repairing peripheral nerve injury.In the application, it is verified through specific examples that adding guanine and isoguanine in resuspended DRG neurons in vitro can significantly promote the growth of DRG neuron axons, and in the application, guanine and isoguanine can regulate DRG neuron cell axon growth in the process of peripheral nerve injury and regeneration, which helps better understand the important role of Guanine and Isoguanine in the process of nerve injury repair, and meanwhile, the effectiveness of the drugs for repairing or treating peripheral nerve injury is verified; and a new target point is provided for treatment of related nervous system diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to application of guanine and isoguanine in preparation of a medicine for treating or repairing peripheral nerve injury. BACKGROUND

[0002] Peripheral nerve injury refers to that a nerve trunk, nerve plexus or nerve ending is damaged due to various reasons (such as trauma, traction, compression, etc.), resulting in abnormal nerve function. Such damage can cause disorders in sensory function, muscle movement function and autonomic nerve function of a patient.

[0003] At present, the treatment methods for peripheral nerve injury mainly include surgical treatment, drug treatment and physical treatment. Surgical treatment is often used in the case of nerve fracture or severe compression, and the nerve function is restored by repairing or reconstructing the damaged nerve. Drug treatment can involve painkillers, anti-inflammatory drugs and neurotrophic drugs, etc. to relieve pain, reduce inflammation and promote nerve function recovery. Physical treatment includes massage, electrical stimulation, warm therapy, etc., aiming to help restore the function of muscles and nerves.

[0004] And the activation of the intrinsic regenerative capacity of neurons after peripheral nerve injury is affected by various factors, including metabolites of the cell body and the surrounding microenvironment after neuronal injury. Seeking key metabolites related to the regeneration of peripheral nervous system neurons will help the regeneration and repair after peripheral nerve injury. SUMMARY

[0005] The present application aims to solve the problem of lack of key metabolites related to the regeneration of peripheral nervous system neurons in the prior art.

[0006] To achieve the above-mentioned purpose, the present application adopts the following technical solutions:

[0007] Application of guanine and / or isoguanine in preparation of a medicine for treating or repairing peripheral nerve injury.

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

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

[0010] The present application also provides a medicine for treating or repairing peripheral nerve injury, which at least comprises guanine and / or isoguanine.

[0011] Preferably, the medicine further comprises other auxiliary medicinal agents acceptable in medicine.

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

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

[0014] (2) In the embodiments of the present invention, it is proposed that adding Guanine or Isoguanine to DRG neurons resuspended in vitro can significantly promote the growth of DRG neuron axons; in the present invention, Guanine and Isoguanine 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 nerve injury repair process, and at the same time verifyes the effectiveness of drugs for repairing or treating peripheral nerve injury; providing new targets for the treatment of related nervous system diseases. Attached Figure Description

[0015] Figure 1 This image shows the cell viability levels of DRG neurons cultured in medium containing different concentrations of Guanine and Isoguanine, as measured using the CCK8 assay in Example 1 of this invention. Different concentrations of Guanine and Isoguanine did not significantly affect the viability levels of DRG neurons.

[0016] Figure 2 This is a diagram illustrating the effect of Guanine or Isoguanine-containing culture medium on the growth of axons in DRG neurons cultured in vitro in Example 1 of the present invention; wherein, Figure 2 A shows the growth of DRG neuron axons after pre-injury treatment with different concentrations of Guanine and Isoguanine, with anti-Tuj1 labeling of 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-damage conditions.

[0017] Figure 3 This is a diagram illustrating the effect of intraperitoneal injection of Guanine and Isoguanine on axonal growth in rats after sciatic nerve injury, as described in Example 2 of the present invention; wherein, Figure 3 A is a flowchart of animal experiments; Figure 3 B shows the growth of axons in rats after sciatic nerve injury detected by immunohistochemistry after intraperitoneal injection of Guanine or Isoguanine, and anti-SCG10 labeled axons of regenerated DRG neurons.Figure 3 C is a statistical graph of fluorescence intensity at different distances from the lesion site based on immunohistochemical results; Figure 3 Figure D shows the statistical results of axonal regeneration coefficient after intraperitoneal injection of Guanine or Isoguanine. Detailed Implementation

[0018] The present invention will be further described in detail below with reference to specific embodiments.

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

[0020] Based on this, this application also provides a drug for treating or repairing peripheral nerve injury, the drug comprising at least guanine and / or isoguanine, and in one embodiment, the drug further comprising other medically acceptable adjuvants.

[0021] The above content will be verified and explained in the following specific verification experiments.

[0022] Example 1: Guanine and Isoguanine in vitro treatment promotes DRG neurite growth

[0023] 1-1 Extraction of primary DRG neurons

[0024] Several adult SD rats were anesthetized with a compound anesthetic. The hair on their backs was shaved, and the rats were disinfected with 75% alcohol. Dissection began from the tail, and the complete spine was removed. The upper lamina was opened, and all DRGs were removed and placed in a dish containing Hibernate A (HA + 1% PS). After all 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 thoroughly minced using ophthalmic scissors. Collagenase was added to the appropriate amount (1 mL for every 3 rats' DRGs), and the dish was placed in a 37°C, 5% CO2 incubator for 90 min of digestion. All liquid in the dish was transferred to a 5 mL agar. Centrifuge at 1200 rpm for 5 min in EP tubes, discard the supernatant, add an equal volume of 0.25% Trypsin-EDTA (preheated at 37°C) to the collagenase, and pipette 10-20 times. Return to a 37°C, 5% CO2 incubator for 3 min, then remove and pipette 10-20 times more until the DRG tissue block is completely digested. Add 3 times the volume of digestion stop solution (10% FBS in PBS) to the trypsin, and pipette 1 mL to mix. Filter all liquid through a 70 μm sieve and collect the cell filtrate. Centrifuge the collected cell filtrate at 1200 rpm for 5 min, discard the supernatant, add 10 mL of 15% BSA, pipette 1 mL to mix, centrifuge at 900 rpm for 5 min, discard the supernatant and any floating or suspended matter, and add 7 mL of... 15% BSA was pipetted and mixed evenly, and centrifuged at 900 rpm for 5 min. The supernatant and floating and suspended matter were discarded. An appropriate amount of adult rat dorsal root ganglion neuron cell (DRG) culture medium (NB+) was added and the purified cells were mixed and evenly seeded into PLL-coated 24-well plates. Finally, the cells were placed in a 37℃, 5% CO2 constant temperature cell culture incubator for culture.

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

[0026] Primary DRG neuronal cells were extracted in step 1 and seeded into 96-well plates. After culturing for 24 hours at 37°C in a 5% CO2 incubator, the original culture medium was discarded. 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, or 1 μM Guanine and Isoguanine, along with their controls (equal volumes of complete culture medium), were added to the cells at 200 μl / well, with three replicates for each treatment. After 24 hours of culture, the culture medium was discarded, and culture medium containing 10% CCK8 reagent was added. The cells were then cultured at 37°C in a 5% CO2 incubator for 2 hours. Cells were harvested, and the absorbance at 450 nm was measured using a microplate reader. Cell viability was also assessed.

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

[0028] 1-3. Primary DRG neuron in vitro injury model

[0029] After culturing neurons in 1-1 for 60-72 hours, discard the original culture medium and add 1 mL of 0.025% trypsin pre-warmed to 37℃ to each well. Place the plate in a cell culture incubator and digest for 3-5 minutes. Add 10% FBS to stop digestion, 2 mL / well. Repeatedly pipette to stop digestion until adherent cells are suspended and basically dispersed into single cells. Transfer the cell suspension from the plate to a sterile 5 mL centrifuge tube and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in an appropriate amount of complete neuronal culture medium, and seed 400 μL / well in pre-coated 24-well plates containing round glass slides, with approximately 5000-6000 cells per well.

[0030] 1-4. Guanine and Isoguanine treat damaged DRG neurons

[0031] After cell resuspending, immediately add 10 nM or 1 μM Guanine or Isoguanine and their controls (equal volume of diluted culture medium) to the cells at 500 μl / well, with 3 replicates for each treatment condition. Observe the axonal growth length of neurons. After 24 h, harvest cells for immunofluorescence assays.

[0032] 1-5. Cell immunofluorescence

[0033] Discard the cell culture medium, gently add 0.5 mL of 1×PBS to wash away the culture medium, and incubate at room temperature for 5 min; discard 1×PBS, add pre-warmed 4% paraformaldehyde, and fix at room temperature for 15 min; discard 4% paraformaldehyde, add 0.5 mL of 1×PBS to wash away the paraformaldehyde, and incubate at room temperature for 5 min / wash, for 2 washes; discard 1×PBS, add 0.5 mL of 0.1% PBST to perforate the membrane, and incubate at room temperature for 10 min; discard 0.1% PBST, add 200 μl of immunohistochemistry blocking buffer per well, and block at room temperature for 1 h; discard the blocking buffer, dilute the primary antibody with immunohistochemistry primary antibody dilution buffer (Beyotime), add the primary antibody anti-Tuj1 (Abcam, ab18207, 1:500), and incubate at 4°C overnight;

[0034] Remove the plate from 4°C and allow it to warm to room temperature for 0.5 hours. Discard the primary antibody, add 1×PBS, and incubate at room temperature for 10 minutes per wash, repeating 3 times. Discard the 1×PBS, protect from light, and dilute the secondary antibody with immunohistochemical secondary antibody dilution buffer (Beyotime). Add the secondary antibody Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor. TM 594 (Thermo Fisher Scientific, A11012, 1:500), incubated at room temperature in the dark for 2 hours; discard the secondary antibody, add 1×PBS, incubate at room temperature, wash 3 times, 10 min / wash; in the dark, pick out the round glass slides containing cells, mount with mounting solution containing DAPI (Beyotime), place in a dry dark box, observe and photograph under a ZEISS upright fluorescence microscope. Observe the growth of the protrusions, photograph and save, and count the length of the longest protrusion in each group and the distribution of protrusion length in each group.

[0035] Different concentrations of guanine and isoguanine were used to treat pre-damaged DRG neurons cultured in vitro. The neuronal cytoskeleton was labeled with anti-Tuj1 antibody (scale bar: 100 μm). The results are as follows: Figure 2 As shown in Figure A; the effects of Guanine and Isoguanine on the total and longest axon lengths of cultured DRG neurons under pre-injury conditions are as follows. Figure 2 As shown in B. Therefore, according to Figure 2 It is evident that, compared with the control group, Guanine and Isoguanine can significantly promote the regeneration of axons in pre-damaged cultured DRG neurons.

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

[0037] Fifteen healthy adult male SD rats, 200±20g each, were randomly divided into three groups of five each. The rats were anesthetized intraperitoneally with a compound anesthetic (0.35mL / 100g). The left hind limb was shaved and disinfected with povidone-iodine. The sciatic nerve in the mid-section of the left femur was surgically exposed. The nerve was separated from the muscle and basement membrane. A special clamping forceps was used to clamp the midpoint of the sciatic nerve segment with a clamping force of 54 N for 30 seconds. Following the clamping (including the day of clamping), the three groups were given daily intraperitoneal injections of 0 / 10μM Guanine / 10μM Soguanine (dissolved in physiological saline) (2mL / kg). The rats were sacrificed on the fourth day (animal experimental procedure, as follows). Figure 3As shown in Figure A), the sciatic nerve on the injured side was removed, embedded in OCT, and frozen sectioned. Primary antibody anti-SCG10 (Novus, NBP1-49461, 1:400) and secondary antibody Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 594 (ThermoFisher Scientific, A11012, 1:500) was used to label regenerating axons of DRG neurons and detect axonal growth after sciatic nerve injury.

[0038] Immunohistochemical staining was used to detect axonal regeneration in rats following sciatic nerve injury after intraperitoneal injection of guanine and isoguanine. Figure 3 As shown in Figure B, anti-SCG10 labels the regenerated axons, with a scale bar of 500 μm; immunohistochemical staining detects the relative fluorescence intensity of tissues at different distances from the injury site relative to the injury site, such as... Figure 3 As shown in Figure C. Statistical results of axonal regeneration coefficients after intraperitoneal injection of Guanine and Isoguanine, as shown in Figure C. Figure 3 As shown in D; Statistical test method: one-way ANOVA. Figure 3 B Figure 3 In C and 3D, immunohistochemical results of axonal SCG10 showed that in vivo administration of Guanine and Isoguanine significantly promoted the regeneration of DRG neuron axons after sciatic nerve injury in rats.

[0039] Therefore, the result is as follows Figure 3 As shown, treatment with Guanine and Isoguanine can significantly promote the regeneration of DRG neuron axons after sciatic nerve compression injury.

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

[0041]

[0042]

[0043] In summary, the application of guanine and isoguanine in the preparation of drugs for treating or repairing peripheral nerve injury, as proposed in this application, demonstrates through specific embodiments that adding guanine and isoguanine to DRG neurons resuspended in vitro can significantly promote the growth of DRG neuron axons. Furthermore, guanine and isoguanine in this application can regulate the growth of DRG neuron axons during peripheral nerve injury and regeneration, contributing to a better understanding of the important roles of guanine and isoguanine in nerve injury repair, while also verifying the effectiveness of drugs for repairing or treating peripheral nerve injury; thus providing new targets for the treatment of related neurological diseases.

Claims

1. The use of guanine and / or isoguanine in the preparation of drugs for the treatment or repair of sciatic nerve injury.

2. The use of guanine and / or isoguanine according to claim 1 in the preparation of drugs for treating or repairing sciatic nerve injury, characterized in that: The drug treats or repairs peripheral nerve damage by promoting the growth of DRG neuron axons.

Images