Verification method for arbutin promoting peripheral nerve injury repair

Abstract

This invention relates to the field of tissue engineering technology, and particularly to a method for verifying the effect of arbutin on the repair of peripheral nerve injury. The invention uses SPF-grade SD rats to construct a sciatic nerve transection injury model. Rats are divided into a model control group and an arbutin group. At 2, 4, and 8 weeks after modeling, arbutin is locally injected into the sciatic nerve transection site in the arbutin group. After intervention, nerve tissue is fixed in 4% paraformaldehyde, automatically dehydrated, and embedded in paraffin to prepare 4μm sections. After dewaxing and hydration, myelin sheath is examined using both Fixed Blue and Nissl staining. Microscopic observation combined with ImageJ software is used to quantitatively analyze myelin sheath morphology and Nissl body number to dynamically verify the repair effect of arbutin. Experimental results show that arbutin can reduce early inflammation of nerve injury and significantly promote myelin sheath repair and nerve tissue regeneration in the later stages.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and in particular to a method for verifying the effect of arbutin on the repair of peripheral nerve injury. Background Technology

[0002] Peripheral nerve injury is a common traumatic and secondary disease in clinical practice. It can be caused by a variety of exogenous and endogenous factors, including trauma, infection, metabolic disorders, and degenerative changes. Clinically, traumatic peripheral nerve injury accounts for 3% of all peripheral nerve injuries. International statistics show that approximately 11 out of every 100,000 people suffer peripheral nerve injury due to accidental trauma. Community surveys indicate that the incidence of neuropathic pain caused by factors such as inflammation is about 10%, and the incidence of secondary nerve injuries, such as diabetic peripheral neuropathy, is as high as 50%. Peripheral nerve injury results in characteristic functional abnormalities in the nerve innervation area, accompanied by varying degrees of nerve damage, which in turn leads to organ dysfunction or impairment, severely affecting the patient's quality of life and easily triggering mental health problems such as anxiety and depression. From a pathological perspective, after peripheral nerve tissue is damaged or severed, the axons within the nerve fibers undergo Wallerian degeneration. The directional migration of regenerative cells such as Schwann cells and macrophages, as well as the regulatory effects of inflammatory factors and growth factors, are the core of mediating axon regeneration, angiogenesis, and nerve tissue repair. If the growth direction of regenerative cells or angiogenesis signals are inhibited, it will directly lead to defects in nerve regeneration and repair.

[0003] Currently, clinical treatment for peripheral nerve injury is mainly divided into two categories: non-surgical treatment and surgical treatment. Non-surgical treatment is based on rehabilitation training and physical therapy, supplemented by oral or local injections of medications. Commonly used medications include corticosteroids, antiepileptic drugs, nucleoside analogs, analgesics, and B vitamin supplements, and are mainly suitable for mild or early peripheral nerve injuries. Surgical treatment includes nerve decompression, nerve anastomosis, and nerve transplantation / transfer, and is suitable for severe injuries such as nerve rupture and avulsion. Although surgery can achieve the connection of nerve structures and promote Schwann cell migration and axonal regeneration, most patients in clinical practice still cannot quickly recover the function of the nerve innervation area after surgery, and postoperative tissue scarring can affect nerve conduction rate, thus limiting the overall repair effect.

[0004] In recent years, natural Chinese herbal extracts and herbal factors have become a hot topic in the field of peripheral nerve regeneration due to their unique biological activities. Multiple studies have confirmed that herbal factors have the potential to promote nerve injury repair in cell culture, in vitro tissue culture and other models, and have shown good prospects in the intervention of common nerve damage such as diabetic neuropathy, providing a new candidate direction for the treatment of peripheral nerve injury. Arbutin, a natural plant component extracted from bearberry leaves, has been shown in existing studies to significantly reduce inflammatory responses induced by inflammatory stimuli and decrease the release of inflammatory mediators in colitis and lung injury models. In neurological studies, arbutin can regulate the expression levels of superoxide dismutase, malondialdehyde, and glutathione peroxidase in mouse brain tissue, enhance the body's endogenous antioxidant capacity, and protect against neurotoxicity. It can alleviate memory impairment, hippocampal oxidative damage, and behavioral impairment and oxidative stress in animal models of Parkinson's disease. In vitro experiments have confirmed that arbutin has anti-inflammatory, antioxidant, and neuroprotective effects on organ dysfunction caused by various nerve injuries, and has great potential to become a candidate traditional Chinese medicine factor for peripheral nerve injury repair.

[0005] However, existing research on the neuroprotective effects of arbutin is mostly focused on in vitro models or central nervous system injuries. Systematic and standardized verification studies on the in vivo repair effects of arbutin on peripheral nerve injuries have not yet been conducted. Furthermore, there is currently no unified standard for verification methods to promote the repair of peripheral nerve injuries using traditional Chinese medicine factors. Existing verification methods are mostly designed for chemical drugs, and suffer from problems such as unclear operating parameters, limited detection methods, and a lack of qualitative and quantitative scientific basis for result interpretation. These methods cannot accurately reflect the true effect of arbutin on peripheral nerve injury repair in vivo, nor can they clearly reveal the time-dependent effects of arbutin at different stages of nerve injury repair. Consequently, the subsequent clinical translation and application of arbutin in the field of peripheral nerve injury repair lack reliable experimental data support.

[0006] In summary, establishing a standardized, reproducible, and accurate verification method for arbutin to promote the repair of peripheral nerve injury, and clarifying the repair effect and related characteristics of arbutin on peripheral nerve injury in vivo, has become an urgent technical problem to be solved in promoting the application of arbutin in the field of peripheral nerve injury repair. It also has important practical significance for improving the verification method system of traditional Chinese medicine factors in the field of nerve injury repair. Summary of the Invention

[0007] The purpose of this invention is to provide a verification method for arbutin to promote the repair of peripheral nerve injury, aiming to solve the problem mentioned in the background art that existing neuroprotective studies on arbutin are mostly focused on in vitro models or central nervous system injuries, and systematic and standardized verification studies on the repair effect of arbutin on peripheral nerve injury at the in vivo level have not yet been carried out.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for verifying the effect of arbutin on the repair of peripheral nerve injury, comprising the following steps:

[0009] 1) Establishment of a rat model of sciatic nerve transection injury: 6-8 week old female SPF grade SD rats were selected. The rats were anesthetized with sodium pentobarbital at a dose of 50 mg / kg. After the skin of the rat thigh was prepared, the operation area was disinfected with povidone-iodine and 75% alcohol. A 1.5 cm incision was made in the skin of the rat leg. The biceps femoris muscle was bluntly dissected at the piriformis muscle to expose the sciatic nerve. The nerve was freed for 5 mm at the anterior trunk of the trigeminal branch of the sciatic nerve. The nerve was clamped to cause injury and then transected to establish a model of sciatic nerve transection injury. The model rats were cultured for 1 week until the model was stable.

[0010] 2) Grouping and drug treatment: The model rats were randomly divided into a control group and an arbutin group. The control group did not receive any drug treatment, while the arbutin group received local injections of arbutin at the sciatic nerve transverse site at 2, 4 and 8 weeks after model establishment.

[0011] 3) Tissue sampling: After drug treatment at each time point in step 2), rats of the corresponding group were sacrificed, and the transverse end of the sciatic nerve and the nearby muscle tissue were excised. The tissues were then soaked and fixed in 4% paraformaldehyde for more than 24 hours.

[0012] 4) Paraffin section preparation: The fixed tissue was dehydrated, embedded in paraffin, sectioned, baked, and dewaxed and hydrated in sequence. Finally, the sections were immersed in PBS buffer and rinsed 3 times for 3 minutes each time. The section thickness was 4 μm, and the baking conditions were 65℃ oven baking for 30 minutes.

[0013] 5) Tissue staining detection: The paraffin sections processed in step 4) were stained with Fixed Blue LFB and Nissl NS, observed and photographed under an inverted microscope, and the myelin morphology, number and distribution of Nissl bodies were quantitatively analyzed and the data were recorded using ImageJ software.

[0014] 6) Results verification: By comparing the staining observation results and Image J quantitative analysis data of the control group and the arbutin group at 2 weeks, 4 weeks and 8 weeks, the repair effect of arbutin on peripheral nerve injury was verified based on the structural integrity of nerve myelin sheath and the trend of Nissl body number.

[0015] Preferably, the specific process of the dewaxing and hydration treatment in step 4) is as follows: the slices after baking are placed in xylene I and xylene II for dewaxing for 30 minutes each; then they are soaked in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, 75% alcohol for 5 minutes, 50% alcohol for 2 minutes, and distilled water for 3 minutes for gradient hydration.

[0016] Preferably, the specific procedure for the myelin sheath LFB staining in step 5) is as follows: add LFB staining agent to the paraffin section, stain for 2 minutes, rinse with running water for 5 minutes, dry the section in an oven for 30 minutes, immerse it in 80% alcohol, 90% alcohol and 95% alcohol for 1 minute each to dehydrate, then place the section in xylene for 5 minutes to clear it, and then mount it with neutral resin for microscopic examination.

[0017] Preferably, the specific procedure for Nissl NS staining in step 5) is as follows: the paraffin sections are dewaxed in xylene I and xylene II for 5 minutes each, then immersed in 80% alcohol, 90% alcohol and 95% alcohol for 1 minute each for dehydration, rinsed with distilled water several times for 3 minutes each time, and then placed in Nissl staining solution and stained in a constant temperature incubator at 37°C for 2 hours. After staining, the sections are rinsed with distilled water and separated by color using ethanol of different concentrations. The sections are then cleared in xylene for 5 minutes, mounted with glycerol gelatin and examined under a microscope.

[0018] Preferably, the SPF-grade SD rats in step 1) are kept under the following conditions: ambient temperature of 25°C, relative humidity of 40-60%, circadian rhythm of 12h each, and free access to food and water. The rat sciatic nerve transection injury model has been approved by the experimental animal ethics committee.

[0019] Preferably, in step 1), before severing the sciatic nerve, the sciatic nerve is fixed to the lower edge of the piriformis muscle 3 mm below the sciatic nerve using 9-0 non-absorbable sutures.

[0020] Preferably, the dehydration in step 4) is completed using a fully automatic tissue dehydrator, the paraffin embedding is completed using a paraffin tissue embedding machine, and the sectioning is completed using a rotary microtome.

[0021] Preferably, the criteria for verifying the results in step 6) are as follows: if the reduction level of Nissl bodies in the arbutin group is significantly lower than that in the control group at 2 weeks (P<0.05), and the number of Nissl bodies is significantly higher than that in the control group at 8 weeks (P<0.05), and the degree of disorder of nerve myelin sheath structure in the arbutin group is lower than that in the control group and the repair speed is faster than that in the control group, then it is determined that arbutin has the effect of promoting the repair of peripheral nerve injury.

[0022] Preferably, when constructing the rat sciatic nerve transection injury model in step 1), the number of rats in each group is not less than 15.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. A standardized, reproducible, and accurate method for verifying the effect of arbutin on peripheral nerve injury repair is provided. The key parameters of the entire process, including model construction, drug intervention, tissue processing, staining detection, and result determination, are clearly defined. The method uses dual detection of myelin sheath solid blue staining and Nissl staining combined with ImageJ software for quantitative analysis, which realizes the unification of morphological qualitative observation and data-driven quantitative analysis. This method completely solves the industry pain points of existing methods for verifying nerve repair by traditional Chinese medicine factors, such as the lack of unified standards, single detection methods, and subjective result determination, and significantly improves the scientificity and reliability of the verification results.

[0025] 2. Through dynamic monitoring at three key time points (2 weeks, 4 weeks, and 8 weeks), combined with a rat sciatic nerve transection model that closely matches clinical trauma pathology, the full range of effects of arbutin on nerve injury—early anti-inflammatory, mid-term repair, and late-term regeneration—can be fully tracked. This accurately reflects the true repair effect of arbutin on peripheral nerve injury in vivo, providing direct and solid experimental evidence for the clinical application of arbutin and the development of nerve repair-related drugs.

[0026] 3. The verification method of this invention has strong versatility. It can not only be used to verify the efficacy of arbutin, but also be extended to the evaluation of peripheral nerve injury repair and drug screening of various natural Chinese herbal extracts and bioactive factors, thus building a universal methodological platform for the field of nerve regeneration. Attached Figure Description

[0027] Figure 1 This invention establishes a rat sciatic nerve transverse model and provides a schematic diagram of tissue sampling.

[0028] Figure 2 This is a representative myelin staining image showing the effect of arbutin of the present invention on the inflammatory state of rat sciatic nerve tissue;

[0029] Figure 3 This is a representative Nissl stain and Nissl body image showing the effect of arbutin of the present invention on the inflammatory state of rat sciatic nerve tissue.

[0030] Figure 4 This is a graph showing the statistical results of the number of Nissl bodies in relation to the effect of arbutin of the present invention on the inflammatory state of the sciatic nerve tissue in rats. Detailed Implementation

[0031] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example: Verification of the effect of arbutin on the repair of peripheral nerve injury

[0033] 1. Laboratory animals

[0034] SPF-grade wild-type SD rats (Sprague Dawleyrats, SD) were purchased from the company and housed in the SPF-grade animal room of the Experimental Animal Center of Jinan University. Housing conditions included: room temperature 25℃, relative humidity 40-60%, 12-hour day / night cycle, and free access to food and water during the period. This animal experiment was reviewed and approved by the Ethics Committee of the Experimental Animal Center of Jinan University.

[0035] 2. The main reagents and consumables are shown in the table below.

[0036] Table 1. Experimental Reagents and Manufacturers

[0037]

[0038]

[0039] 3. Major instruments and equipment are shown in the table below.

[0040] Table 2. Main Experimental Instruments and Equipment and Their Manufacturers

[0041]

[0042] 4. Experimental Methods

[0043] 4.1 Rat sciatic nerve transection model

[0044] Thirty female SD rats aged 6-8 weeks were selected and divided into two groups (control group and arbutin group), with 15 rats in each group. Rats were anesthetized intraperitoneally with sodium pentobarbital at a dose of 50 mg / kg. The rat thighs were prepared and disinfected with iodine and 75% alcohol. An incision of approximately 1.5 cm was made in the rat leg skin using ophthalmic scissors. The biceps femoris muscle was bluntly dissected at the piriformis muscle location to expose the sciatic nerve. The sciatic nerve was gently dissected with forceps, and approximately 5 mm of the nerve was freed anterior to the trigeminal branch of the sciatic nerve. The nerve position was fixed with 9-0 non-absorbable sutures. The nerve was first clamped approximately 3 mm below the piriformis muscle to confirm the extent of nerve damage before transection, thus establishing a sciatic nerve transection injury model. The model stabilized after one week, and subsequent grouping and drug treatment were initiated. During the 2, 4, and 8 weeks of sciatic nerve transection model establishment, sciatic nerve transection ends and nearby muscle tissue were collected from the rats at each time point.

[0045] 4.2 Tissue sampling and paraffin sectioning

[0046] (1) Tissue sampling: Rats were sacrificed according to the experimental time period, and muscle and nerve tissues were cut from the transverse model location and soaked in 4% paraformaldehyde for more than 24 hours;

[0047] (2) Dehydration of tissue samples: After soaking the samples for a sufficient time, dehydration is carried out using a fully automated tissue dehydrator;

[0048] (3) Sample embedding: The sample was embedded in paraffin using a paraffin tissue embedding machine;

[0049] (4) Sectioning: After being embedded in paraffin, the tissue was cut into 4μm thick sections using a rotary microtome to prepare paraffin tissue sections;

[0050] (5) Baking: Baking paraffin slices in an oven at 65℃ for 30 minutes;

[0051] (6) Dewaxing and hydration: The sections were dewaxed in xylene (xylene I for 30 min, xylene II for 30 min), and then hydrated (anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 75% ethanol for 5 min, 50% ethanol for 2 min, distilled water for 3 min). Finally, they were immersed in PBS for 3 min each time, for a total of 3 times.

[0052] (7) Staining: Select the staining method according to the experimental purpose and requirements, and mount the slide with neutral resin after following the steps;

[0053] (8) Observation: Observe and take pictures under a microscope.

[0054] 4.3 Myelin staining / Fixol Fast Blue Staining (LFB)

[0055] The Fix blue staining method can stain the myelin sheath within nerve fibers a deep blue color, which can be used to observe differences in myelin sheath tissue morphology. The steps are as follows:

[0056] (1) Staining: Perform LFB staining of sciatic nerve tissue according to the kit instructions. Add Black Blue staining agent to the above sections, stain for 2 min, and rinse with running water for 5 min;

[0057] (2) Drying: Dry in an oven for 30 minutes;

[0058] (3) Gradient dehydration: Immerse the tissue in a gradient of ethanol (80% alcohol, 90% alcohol and 95% alcohol) for 1 min each;

[0059] (4) Clearing and mounting: Clear sections with xylene for 5 min, mount with neutral resin, and cover the sections with a coverslip at an angle to remove air;

[0060] (5) Observation and statistics: Observe and take pictures under an inverted microscope. Measure and record the morphology of myelin sheath.

[0061] 4.4 Nissl staining / Toluidine blue staining (NS)

[0062] Nissl staining stains Nissl bodies in nerve tissue blue (purple), reflecting the degree of damage within the nerve tissue. The number of Nissl bodies can be compared to observe the degree of inflammation, degeneration, and damage in nerve tissue. The steps are as follows:

[0063] (1) Dewaxing of sections: Dewaxing with xylene I and II for 5 minutes each;

[0064] (2) Gradient dehydration: tissues were dehydrated by gradient ethanol (80% alcohol, 90% alcohol, 95% alcohol) for 1 min each;

[0065] (3) Wash the sections: Wash the sections with distilled water about once, for 3 minutes each time;

[0066] (4) Staining sections: The staining solution was placed in a 37°C constant temperature incubator for 2 hours for staining. After soaking, the sections were rinsed with distilled water and then separated by different concentrations of ethanol.

[0067] (4) Clearing and mounting: Clear sections with xylene for 5 min, mount with glycerol gelatin, cover the sections with a coverslip at an angle, and remove all air;

[0068] (5) Observation and statistics: Observe and photograph under an inverted microscope, measure the distribution of Nissl bodies in nerve tissue using Image J, and record and perform statistical analysis.

[0069] 5. Experimental Results

[0070] 5.1 Construction of a rat sciatic nerve transection model and tissue sampling

[0071] like Figure 1 The images, arranged from left to right, show: during the procedure to establish a rat sciatic nerve transect model, after the rat model was established, and the location of sciatic nerve tissue sampling.

[0072] 5.2 The repairing effect of arbutin on sciatic nerve tissue injury

[0073] Sciatic nerve tissue was established in SD rats using a transect model. Arbutin was injected locally at 2, 4, and 8 weeks. Rats were then sacrificed, and samples were prepared, including paraffin embedding and sectioning. LFB staining was used in this study. Figure 2 ) and Nissl staining ( Figure 3The study observed the structure of nerve tissue and the inflammatory process. Results showed that Nissl bodies were significantly reduced in the control group within two weeks of nerve injury (P<0.05), while the reduction in Nissl bodies in the local arbutin injection model group was lower than that in the model group (P<0.05). There were no statistically significant differences in the number of Nissl bodies among the control group, surgical group, and arbutin group at week 4. At week 8, the number of Nissl bodies in the surgical group and arbutin group was higher than that in the control group (P<0.05). LFB staining showed that nerve fibers and cells were intact in the control group, while in the model group and arbutin model group, nerve injury in the first two weeks led to structural disorder in cell bodies and axons (red arrows). Nerve repair was in progress at week 4, with some normally arranged nerve cells visible. Nerve repair was largely completed at week 8, but there was still a possibility of connection or anastomosis with adjacent soft tissue; further experimental measurements are needed to determine the functional recovery status.

[0074] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for verifying the effect of arbutin on the repair of peripheral nerve injury, characterized in that, Includes the following steps: 1) Establishment of a rat model of sciatic nerve transection injury: 6-8 week old female SPF grade SD rats were selected. The rats were anesthetized with sodium pentobarbital at a dose of 50 mg / kg. After the skin of the rat thigh was prepared, the operation area was disinfected with povidone-iodine and 75% alcohol. A 1.5 cm incision was made in the skin of the rat leg. The biceps femoris muscle was bluntly dissected at the piriformis muscle to expose the sciatic nerve. The nerve was freed for 5 mm at the anterior trunk of the trigeminal branch of the sciatic nerve. The nerve was clamped to cause injury and then transected to establish a model of sciatic nerve transection injury. The model rats were cultured for 1 week until the model was stable. 2) Grouping and drug treatment: The model rats were randomly divided into a control group and an arbutin group. The control group did not receive any drug treatment, while the arbutin group received local injections of arbutin at the sciatic nerve transverse site at 2, 4 and 8 weeks after model establishment. 3) Tissue sampling: After drug treatment at each time point in step 2), rats of the corresponding group were sacrificed, and the transverse end of the sciatic nerve and the nearby muscle tissue were excised. The tissues were then soaked and fixed in 4% paraformaldehyde for more than 24 hours. 4) Paraffin section preparation: The fixed tissue was dehydrated, embedded in paraffin, sectioned, baked, and dewaxed and hydrated in sequence. Finally, the sections were immersed in PBS buffer and rinsed 3 times for 3 min each time. The section thickness was 4 μm, and the baking conditions were 65℃ oven baking for 30 min. 5) Tissue staining detection: The paraffin sections processed in step 4) were stained with Fixed Blue LFB and Nissl NS, observed and photographed under an inverted microscope, and the myelin morphology, number and distribution of Nissl bodies were quantitatively analyzed and the data were recorded using ImageJ software. 6) Results verification: By comparing the staining observation results and Image J quantitative analysis data of the control group and the arbutin group at 2 weeks, 4 weeks and 8 weeks, the repair effect of arbutin on peripheral nerve injury was verified based on the structural integrity of nerve myelin sheath and the trend of Nissl body number. 2.The method of claim 1, wherein the method is characterized by, The specific process of dewaxing and hydration in step 4) is as follows: after baking, the slices are placed in xylene I and xylene II for dewaxing for 30 minutes each; then they are soaked in anhydrous ethanol I and anhydrous ethanol II for 5 minutes each, 75% alcohol for 5 minutes, 50% alcohol for 2 minutes, and distilled water for 3 minutes for gradient hydration. 3.The method of claim 1, wherein the method is characterized by, The specific procedure for myelin fix blue LFB staining in step 5) is as follows: Add fix blue staining agent to the paraffin section, stain for 2 minutes, rinse with running water for 5 minutes, dry the section in an oven for 30 minutes, immerse it in 80% alcohol, 90% alcohol and 95% alcohol for 1 minute each to dehydrate, then place the section in xylene for 5 minutes to clear it, mount it with neutral resin and examine it under a microscope. 4.The method of claim 1, wherein the method is characterized by, The specific procedure for Nissl NS staining described in step 5) is as follows: Paraffin sections are dewaxed in xylene I and xylene II for 5 minutes each, then immersed in 80% alcohol, 90% alcohol and 95% alcohol for 1 minute each for dehydration, and rinsed with distilled water several times for 3 minutes each time; the sections are then placed in Nissl staining solution and soaked in a 37°C constant temperature incubator for 2 hours for staining. After staining, the sections are rinsed with distilled water, separated by different concentrations of ethanol, and then placed in xylene for 5 minutes for clearing. After mounting with glycerol gelatin, the sections are examined under a microscope.

5. The method of claim 1, wherein the method is for verifying the promotion of peripheral nerve injury repair by arbutin. The SPF-grade SD rats described in step 1) were kept under the following conditions: ambient temperature of 25°C, relative humidity of 40-60%, circadian rhythm of 12h each, and free access to food and water. The rat sciatic nerve transection injury model was constructed and approved by the experimental animal ethics committee. 6.The method of claim 1, wherein the method is characterized by, In step 1), before sciatic nerve transect, the sciatic nerve is fixed 3 mm below the piriformis muscle using 9-0 non-absorbable sutures.

7. The verification method for arbutin promoting the repair of peripheral nerve injury according to claim 1, characterized in that, The dehydration in step 4) is completed using a fully automatic tissue dehydrator, the paraffin embedding is completed using a paraffin tissue embedding machine, and the sectioning is completed using a rotary microtome.

8. The verification method for arbutin promoting the repair of peripheral nerve injury according to claim 1, characterized in that, The criteria for verifying the results described in step 6) are as follows: if the reduction level of Nissl bodies in the arbutin group is significantly lower than that in the control group at 2 weeks (P<0.05), and the number of Nissl bodies is significantly higher than that in the control group at 8 weeks (P<0.05), and the degree of disorder of nerve myelin sheath structure in the arbutin group is lower than that in the control group and the repair speed is faster than that in the control group, then it is determined that arbutin has the effect of promoting the repair of peripheral nerve injury. 9.The method of claim 1, wherein the method is characterized by, When constructing the rat sciatic nerve transection injury model in step 1), the number of rats in each group should not be less than 15.

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