Application of nucleic acid binding protein ZBP1 in treatment of brachial plexus root avulsion injury

Abstract

The invention relates to the field of medicine, in particular to application of nucleic acid binding protein ZBP1 in treatment of brachial plexus root avulsion injury. Brachial plexus root avulsion and replantation operations are respectively carried out on a WT mouse and a ZBP1 gene knockout mouse, and it is confirmed that ZBP1 gene knockout can promote motor function recovery, reduce motor neuron death, increase myelin regeneration and relieve muscle atrophy. Meanwhile, the ZBP1 gene knockout can reduce the death of motor neurons and promote the recovery of motor functions. The invention aims to provide a novel treatment strategy and a medicine development and research direction for treating brachial plexus root avulsion injury.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of nucleic acid binding protein ZBP1 in treating brachial plexus root avulsion injury. Background Art

[0002] Brachial plexus injury is a common type of peripheral nerve injury, primarily manifesting as motor dysfunction, paresthesias, areflexias, and muscle atrophy in the shoulder, arm, wrist, and hand. Brachial plexus root avulsion (BPA), the most severe form of upper limb peripheral nerve injury, occurs when spinal nerve roots on the surface of the spinal cord are avulsed, resulting in a disconnection between the neuronal cell bodies and target muscle fibers. This injury can induce degeneration and death of most motor neurons, axonal degeneration, and atrophy of the muscles controlled by the corresponding spinal cord segment, resulting in unilateral or bilateral paralysis. Furthermore, BPA can induce severe neuropathic pain, which is caused by abnormal activity in non-afferent spinal cord segments. There is a correlation between the number of root avulsions and pain severity. Although renervation surgery is available clinically, the slow growth of axons in the anterior horn motor neurons prevents reinnervation of the biceps brachii before the target muscle atrophies, making complete recovery of motor function difficult. Motor neuron survival is crucial for functional recovery of the affected limb, while inflammation and apoptosis in the early stages of brachial plexus root avulsion can lead to the death of a large number of neurons.

[0003] ZBP1 (Z-DNA binding protein 1) is a Z-DNA and Z-RNA binding protein. It is a natural receptor that recognizes Z-DNA / RNA produced after viral infection and can trigger different forms of cell death and inflammatory responses. It plays an important role in cell apoptosis, necrosis and inflammatory responses.

[0004] There is currently no existing technology to study the association between ZBP1 and brachial plexus root avulsion injury. Summary of the Invention

[0005] The purpose of the present invention is to provide an application of nucleic acid binding protein ZBP1 in treating brachial plexus root avulsion injury.

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

[0007] Application of nucleic acid binding protein ZBP1 in the preparation of a drug for treating brachial plexus root avulsion injury.

[0008] In one preferred embodiment, the application is the use of the nucleic acid binding protein ZBP1 in the preparation of a drug for promoting nerve regeneration and repair after brachial plexus root avulsion surgery.

[0009] In one preferred embodiment, the surgery for treating brachial plexus nerve root avulsion injury includes nerve root reimplantation.

[0010] This study used a brachial plexus root avulsion injury model in WT mice and ZBP1 knockout mice to determine the effects of ZBP1 deficiency on brachial plexus root avulsion injury. The results showed that ZBP1 knockout promoted motor function recovery, reduced motor neuron death, increased myelin regeneration, and alleviated muscle atrophy.

[0011] Based on the same inventive concept, the present invention also claims protection for the use of an agent for knocking out the ZBP1 gene or an agent for reducing the level of the ZBP1 gene in the preparation of a drug for treating brachial plexus root avulsion injury.

[0012] In a preferred embodiment, the reagent for knocking out the ZBP1 gene is CRISPR-Cas9.

[0013] In one preferred embodiment, the application is the use of an agent for knocking out the ZBP1 gene or an agent for reducing the level of the ZBP1 gene in the preparation of a drug for promoting nerve regeneration and repair after brachial plexus root avulsion surgery.

[0014] In one preferred embodiment, the surgery for treating brachial plexus nerve root avulsion injury includes nerve root reimplantation.

[0015] In one preferred embodiment, the reagent for knocking out the ZBP1 gene includes: Cas9 nuclease (Thermo Fisher, TrueCut TM HiFi Cas9 protein), sgRNA that guides Cas9 to the ZBP1 gene, a transfection reagent comprising a Cas9 nuclease that specifically cuts the ZBP1 gene and an sgRNA that guides Cas9 to the ZBP1 gene, a TALEN protein targeting the ZBP1 gene, a vector or transfection reagent comprising a TALEN protein targeting the ZBP1 gene, siRNA that can specifically bind to and degrade ZBP1 gene mRNA, and a transfection reagent comprising siRNA that specifically binds to and degrades ZBP1 gene mRNA.

[0016] In one preferred embodiment, the Cas9 nuclease that specifically cuts the ZBP1 gene is ThermoFisher, TrueCut TM HiFi Cas9 protein.

[0017] In one preferred embodiment, the transfection reagent comprising the Cas9 nuclease that specifically cuts the ZBP1 gene and the sgRNA that guides Cas9 to the ZBP1 gene is Lipofectamine 3000 transfection reagent, purchased from Thermo Fisher Scientific.

[0018] In one preferred embodiment, the vector containing the TALEN protein targeting the ZBP1 gene is a pCS2-TALEN vector.

[0019] In one preferred embodiment, the transfection reagent containing siRNA that specifically binds to and degrades ZBP1 gene mRNA is DharmaFECT transfection reagent, purchased from Horizon Discovery.

[0020] In one preferred embodiment, the reagent for reducing the level of ZBP1 gene includes: siRNA that can complementally pair with a specific sequence of ZBP1 gene mRNA, a transfection reagent containing siRNA that can complementally pair with a specific sequence of ZBP1 gene mRNA, a kit containing siRNA that can complementally pair with a specific sequence of ZBP1 gene mRNA, miRNA targeting ZBP1 mRNA, a short single-stranded DNA or RNA molecule complementary to ZBP1 mRNA, an inactivated Cas9 that binds to the promoter or enhancer region of ZBP1 gene, a Cas9 nuclease that specifically cuts ZBP1 gene, an sgRNA that guides Cas9 to ZBP1 gene, a transfection reagent containing a Cas9 nuclease that specifically cuts ZBP1 gene and an sgRNA that guides Cas9 to ZBP1 gene, a TALEN protein targeting ZBP1 gene, a vector or transfection reagent containing a TALEN protein targeting ZBP1 gene, siRNA that can specifically bind to and degrade ZBP1 gene mRNA, and a transfection reagent containing siRNA that specifically binds to and degrades ZBP1 gene mRNA.

[0021] In one preferred embodiment, the kit containing siRNA that can complementally pair with a specific sequence of ZBP1 gene mRNA is purchased from Sigma-Aldrich.

[0022] In one preferred embodiment, the Cas9 nuclease that specifically cuts the ZBP1 gene is the TrueCut Cas9 protein from ThermoFisher, the sequence of the sgRNA that guides Cas9 to the ZBP1 gene is: 5'-GGAGGATTGCTATGAGTTCCAGG-3', the transfection reagent containing the Cas9 nuclease that specifically cuts the ZBP1 gene and the sgRNA that guides Cas9 to the ZBP1 gene is purchased from Thermo Fisher, the TALEN protein targeting the ZBP1 gene is from Cellectis, the vector or transfection reagent containing the TALEN protein targeting the ZBP1 gene is from Addgene, the siRNA that can specifically bind to and degrade the ZBP1 gene mRNA is purchased from Thermo Fisher, and the transfection reagent containing the siRNA that specifically binds to and degrades the ZBP1 gene mRNA is from Thermo Fisher, Lipofectamine TM RNAiMAX.

[0023] The present invention has been demonstrated through experiments that the number of motor neurons in mice after ZBP1 gene knockout is significantly increased, the abnormal morphology of musculocutaneous nerve fibers is improved, the neuronal death of the brachial plexus nerves in mice after injury and replantation is reduced, the expression of myelination indicators NGFR and POU3F1 is reduced, and the spinal cord myelination in mice after brachial plexus nerve root avulsion and replantation is reduced, indicating that ZBP1 gene knockout improves the survival of motor neurons in brachial plexus nerve root avulsion and replantation, and promotes the recovery of motor function.

[0024] In one preferred embodiment, the drug further comprises one or more pharmaceutically acceptable carriers, including coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH regulators, and flavoring agents.

[0025] In a preferred embodiment, the dosage form of the drug includes any one of tablets, capsules, granules, powders, powder injections, pills, emulsions, suspensions, and solutions.

[0026] In a preferred embodiment, the administration of the drug includes any one of injection, oral administration, absorption, physical or chemical mediation to introduce the drug into the body; or the drug is mixed or encapsulated with other substances and then introduced into the body.

[0027] In a preferred embodiment, the injection includes any one of intravenous injection, intramuscular injection, and mucosal tissue injection.

[0028] This study, conducted by performing brachial plexus root avulsion and reimplantation surgery on WT and ZBP1 knockout mice, confirmed that ZBP1 knockout promoted motor function recovery, reduced motor neuron death, increased myelin regeneration, and alleviated muscle atrophy. Furthermore, ZBP1 knockout reduced motor neuron death and promoted motor function recovery. This study aims to provide a new treatment strategy and drug development research direction for treating brachial plexus root avulsion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The expression level of Zbp1 mRNA in the anterior horn of the spinal cord of mice after BPRA.

[0030] Figure 2 Figure 3: ZBP1 gene knockout promotes motor function recovery in BPRA mice. Figure A shows the scoring criteria for the face-washing test, Figure B shows the results of the face-washing test, and Figure C shows the results of the cylinder test.

[0031] Figure 3 Figure 1 shows that ZBP1 gene knockout reduces neuronal death in mice after BPRA. Figure A shows the results of neutral red staining, Figure B shows the results of Bax in ZBP1 gene knockout mice, and Figure C shows the results of caspase-3 in ZBP1 gene knockout mice.

[0032] Figure 4 Figure 1: ZBP1 knockout inhibits pyroptosis and inflammation in spinal cord cells of BPRA mice. Figure A shows the results of caspase-4 in spinal cord cells of ZBP1 knockout mice. Figure B shows the results of GSDMD in spinal cord cells of ZBP1 knockout mice. Figure C shows the results of NLRP3 in spinal cord cells of ZBP1 knockout mice. Figure D shows the results of IL-6, an inflammatory indicator in spinal cord cells of ZBP1 knockout mice. Figure E shows the results of TNF-α in spinal cord cells of ZBP1 knockout mice.

[0033] Figure 5 Figure 1: Knockout of ZBP1 promotes remyelination of nerve fibers in BPRA mice. Figure A shows the results of LFB staining experiments; Figure B shows the results of Chat immunofluorescence experiments; Figure C shows the expression results of ngfr and pou3f1, the myelination indicators, by qRT-PCR.

[0034] Figure 6 ZBP1 gene knockout reduces muscle atrophy in BPRA mice; Figure A shows the experimental results of mouse muscle weight; Figure B shows the results of H&E experiment. DETAILED DESCRIPTION

[0035] The present invention is not limited to the following specific embodiments. Based on the disclosure of the present invention, a person skilled in the art may adopt a variety of other specific embodiments to implement the present invention. Any simple changes or modifications made to the design structure and concept of the present invention fall within the scope of protection of the present invention. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other unless they conflict.

[0036] Example 1

[0037] Zbp1 mRNA expression in the anterior horn of the spinal cord after brachial plexus root avulsion and reimplantation in WT mice.

[0038] (1) Materials and methods

[0039] 1. Experimental grouping and modeling

[0040] In this experiment, WT mice (Nanjing Jicui Yaokang Biological Co., Ltd., 8 weeks old, 7 mice) were used as control mice. Male and female mice were separated into cages and adaptively fed for 1 week. Before using surgical surgery to establish the experimental animal model, carefully check whether the right forelimb of each mouse has movement disorders, weight, health status, etc., and try to ensure the uniformity of the experimental animals. A 1.25% tribromoethanol anesthetic was injected intraperitoneally at a concentration of 20 μl / g to put the animal into deep anesthesia. After removing the hair on the neck with electric clippers, the experimental animal was placed flat on a sterile surgical pad. Iodine was used to disinfect the surgical site on the back. In order to protect the mouse's eyes from damage caused by the light during surgery, erythromycin ointment was applied to its left and right eyes respectively. Under a dissecting microscope, at the midline of the neck, the skin of the mouse's neck was gently cut with ophthalmic scissors. Then, a surgical retractor (homemade, sterilized) was used to separate the fat on both sides of the neck, and excess muscle and fascia were removed with a scalpel. In the exposed surgical field of view, the T2 spinous process is easily identified. Based on its segmental location, the three spinal cord segments requiring avulsion (C5, C6, and C7) are precisely determined. The vertebral arches and superior articular processes of the C4-7 segments on the right side of the spinal cord are then surgically resected. After tearing open the spinal dura mater, the dorsal nerve roots of C5-C7 are precisely isolated. The ventral roots are then exposed separately, and any bleeding is stopped with a medical absorbable sponge. After local anesthesia with lidocaine, the right ventral roots of C5 and C7 are torn with forceps and completely removed, then cut to equal lengths. Using the same method as above, the C6 ventral root is removed and reimplanted into its original spinal cord segment location. The muscle fascia and skin are carefully sutured. The dorsal suture site is then disinfected with iodine. After completion of the above procedures, the animals are kept warm and awake to establish a brachial plexus root avulsion-reimplantation model. All rats receive routine analgesic and anti-infective medications postoperatively. All animals were returned to their cages after waking up to ensure their normal life.

[0041] 24 hours after surgery, the rats were evaluated for motor function for the first time. The rats were evenly sprayed with approximately equal amounts of sterile DDW from multiple directions on their heads and then placed in a cylindrical glass cup. A camera was used to record a video for 5 minutes to observe the position of the forepaws reaching the head when the rats were forced to bend their elbows to remove water droplets from their heads. Figure 2 The scoring criteria for the face-washing test listed in A are based on a 0-5 rating scale (0: unable to flex the elbow, 0 points; 1: elbow flexed but unable to touch the nose, 1 point; 2: nose can be touched at the highest point of elbow flexion, 2 points; 3: below the eyes can be touched, 3 points; 4: eyes can be touched, 4 points; 5: forelimbs raised to touch / above the ears, 5 points). The highest motor score within 5 minutes is recorded. The score ranges from 0 to 5, with higher scores indicating better motor function recovery. Animals with successful surgery are scored as 0; animals with scores greater than 0 are eliminated. This ensures successful modeling.

[0042] 2. Tissue Preparation: Eight weeks post-operatively, mice were anesthetized with an intraperitoneal injection of tribromoethanol. Once deeply anesthetized, the chest cavity was opened with surgical instruments, the heart exposed, and a needle was inserted into the left ventricle quasi-apically. The right atrial appendage was gently cut open. PBS was first perfused, followed by 4% PFA fixative, until the liver turned pink and the body became rigid. The required C5-C7 spinal cord tissue, bilateral musculocutaneous nerves, and bilateral biceps brachii were removed using surgical instruments. The biceps brachii tissue was weighed first. The tissues were then fixed in 4% PFA overnight and dehydrated in 10%, 20%, and 30% sucrose solutions, respectively, until they sank to the bottom. The precipitated tissues were removed, embedded in OCT, and stored at -80°C until further use. The tissues were removed from the -80°C freezer and pre-cooled in a -20°C cryostat for 30 minutes to match the temperature inside the microtome. Fix the tissue in OCT on a steel ice tray in a microtome. After fixation, adjust the microtome and perform serial sectioning at a thickness of 8 μm. Store the sections in a -80°C freezer until needed. Alternatively, after perfusing fresh tissue with PBS, remove the desired tissue and place it in an enzyme-free EP tube. Store the tissue in a -80°C freezer until needed.

[0043] 3. Real-time fluorescence quantitative PCR

[0044] 1) RNA extraction: Fresh mouse spinal cord tissue was obtained, 1 ml of RNA extraction reagent Trizol (Sevier, G3010) was added, and the tissue was ground with a grinding rod to extract total RNA. 200 ml of chloroform substitute (Sevier, G3014) was added and the tube was thoroughly mixed by vortexing. The tube was allowed to stand at room temperature for 5 min, and centrifuged at 4°C and 12,000 rpm for 10 min. The upper clear aqueous phase was carefully pipetted into a new 1.5 ml EP tube, and an equal volume of isopropanol was added. The tube was mixed by inversion. The tube was allowed to stand at room temperature for 5 min, and centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was discarded, 1 ml of 75% ethanol was added, and the tube was centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was discarded, and the tube was dried at room temperature for 10 min. DEPC water (Sevier, G3004) was added to dissolve the RNA, and the RNA concentration was measured.

[0045] 2) Reverse transcription of cDNA: The RNA in the tissues was reverse transcribed using the 1st Strand cDNA Synthesis SuperMix for qPCR Kit (Yisheng Biotechnology, 11141ES60).

[0046] 3) Real-time fluorescence quantitative PCR detection of gene expression: The reaction system (1 μl cDNA + 0.5 μl 10M forward primer + 0.5 μl 10M reverse primer + 5 μl SYBR Green Mix + 5 μl RNase-free ddH2O) was added to eight PCR tubes and centrifuged. Detection was performed using a qRT-PCR instrument using a two-step protocol (95°C, 5 min; 95°C, 10 sec; 60°C, 30 sec; 40 cycles, with a melting curve program). qRT-PCR primers were designed and prepared by Shanghai Sangon Biotechnology Co., Ltd. The sequences of qRT-PCR primers are shown in Table 1. The expression of zbp1 mRNA in the anterior horn of the spinal cord was assayed.

[0047] Table 1 qRT-PCR amplification primers

[0048]

[0049] (2) Experimental results

[0050] 1. Expression of zbp1 mRNA in the anterior horn of the spinal cord after brachial plexus root avulsion and reimplantation in WT mice. Figure 1As shown, Zbp1 mRNA expression in the anterior horn of the spinal cord of mice after BPRA increased. These experimental results indicate that Zbp1 mRNA expression in the anterior horn of the spinal cord of mice after BPRA increases, and knocking out the ZBP1 gene may be beneficial for brachial plexus nerve recovery.

[0051] Example 2

[0052] Construction of ZBP1 gene knockout mice

[0053] ZBP1 gene knockout mice were purchased from Jiangsu Jicui Pharmaceutical, No. T029037. The ZBP-1 gene (Gene ID: 58203) was knocked out using CRISPR-Cas9 technology: a highly specific sgRNA was designed targeting exons 2-8 of the ZBP1 gene (corresponding to transcript ZBP1-201, containing 1202 bp encoding the key protein domain, ENSMUST00000029018.13), and Cas9 was synthesized by in vitro transcription using T7 RNA polymerase. mRNA (100 ng / μL) and sgRNA (50 ng / μL), and the mixture (1-2 pL) was injected into the male pronucleus of C57BL / 6J mouse fertilized eggs using microinjection technology. After culture to the blastocyst stage, it was transplanted into the uterus of pseudopregnant female mice; F0 generation mice were breastfed after birth, and DNA was extracted by tail clipping after 3 weeks of feeding. DNA extraction was as follows: mouse toes were placed in a 1.5 ml EP tube, 100 ul (50 mM) NaOH was added to each tube, and rapid shaking was performed; 95℃-100℃ metal bath for 30 minutes; 10 ul (1 M; Tris PH = 7.0) was added to each tube for neutralization, vortexed and shaken, and centrifuged at 15000 rpm for 2 minutes for later use.

[0054] The extracted DNA was amplified by PCR using two pairs of primers: PCR ①: F1 / R1, expected wild-type 12128 bp / KO-type 315 bp; PCR ②: F2 / R2, expected wild-type 251 bp / KO-type 0 bp.

[0055] The PCR amplification primer sequences are as follows:

[0056] Table 2 PCR amplification primers

[0057]

[0058]

[0059] PCR system construction: PCR amplification was performed according to the Vazyme-P222 kit: 2× Rapid Taq Master Mix) 12.5 μl + ddH2O 9.5 μl + Primer F (10 pmol / μl) 1 μl + Primer R (10 pmol / μl) 1 μl + 1 μl DNA.

[0060] PCR program: 95°C, 3 min; 95°C, 30 s, 65°C, 30 s, 72°C, 30 s (20 cycles); 95°C, 30 s, 55°C, 30 s, 72°C, 30 s (15 cycles); 72°C, 5 min; 10°C, hold.

[0061] PCR amplification products were verified by gel electrophoresis as follows: 1.4 g of agarose was weighed and placed in a conical flask. 70 ml of 1× TAE was added and shaken by hand. Microwaved to a first boil, removed and shaken, returned to the microwave, brought to a second boil, removed and shaken, returned to the microwave, and brought to a third boil, removed and shaken. Cooled to approximately 55°C, 1 / 10,000 nucleic acid dye was added, shaken, and slowly applied to the gel plate. Allow to stand at room temperature until the gel was completely solidified. The sample was then added to the gel wells and heated at 140 V, 40 mA, for 30 minutes.

[0062] Positive individuals were screened and backcrossed with the wild type to establish F1 heterozygotes, and finally homozygous knockout lines were obtained by intercrossing the F1 heterozygotes.

[0063] The reagents involved are: Cas9 nuclease that specifically cuts the ZBP1 gene (Thermo Fisher, TrueCutCas9 protein), sgRNA that guides Cas9 to the ZBP1 gene, a transfection reagent containing Cas9 nuclease that specifically cuts the ZBP1 gene and sgRNA that guides Cas9 to the ZBP1 gene (Thermo Fisher), TALEN protein for the ZBP1 gene (Cellectis), a vector or transfection reagent containing TALEN protein for the ZBP1 gene (Addgene), siRNA that can specifically bind to and degrade ZBP1 gene mRNA (Thermo Fisher), a transfection reagent containing siRNA that specifically binds to and degrades ZBP1 gene mRNA (Thermo Fisher, Lipofectamine TM RNAiMAX). ZBP1 gene-specific primers were synthesized by Shanghai Sangon Synthetic Engineering Co., Ltd. The sequences are shown in Table 1. Agarose (Sangong, A620014-0100) was used.

[0064] 5) Result analysis:

[0065] PCR ① detects the knockout region itself (12128bp→315bp) to confirm whether exons 2-8 are deleted. PCR ② verifies whether the downstream of the knockout region (exon 9 and beyond) remains intact to rule out off-target effects.

[0066] The results of PCR verification are as follows: the sample shows a 315 bp band in PCR ①, and no band in PCR ② → confirming successful gene knockout.

[0067] Example 3

[0068] Effect of ZBP1 deficiency on the recovery of motor function in mice after brachial plexus root avulsion and reimplantation surgery

[0069] (1) Materials and methods

[0070] 1. Experimental grouping, modeling, and tissue preparation: The control mice, namely wild-type mice, and the ZBP1 gene knockout group mice were subjected to modeling and tissue preparation, and the specific process was the same as in Example 1.

[0071] 2. Motor function test

[0072] 2.1 Face washing experiment

[0073] The recovery of the right forelimb motor function of the mice was checked regularly once a week. All animal behavior experiments were conducted at the same time in the morning. The double-blind method was used to score the experimental animal behavior. Almost equal amounts of sterile DDW were evenly sprayed on the head of the mouse from multiple directions, and then the mouse was placed in a cylindrical glass cup. A camera was used to shoot a video for 5 minutes to observe the position of the forepaws reaching the head when the mouse was forced to bend its left and right forelimbs to remove water droplets from its head. According to Figure 2 The face-washing test scoring criteria listed in A were used, with the highest score within 5 minutes recorded. Testing was conducted weekly for eight weeks post-surgery.

[0074] 3.2 Cylinder experiment

[0075] The cylinder test assesses forelimb motor function recovery. Using a double-blind method, mice are placed in a 50 cm diameter, 70 cm high glass cylinder. Video recording is used to record the number of times the right forepaw touches the cylinder wall until the left forepaw touches the cylinder 20 times. Testing is performed weekly for eight weeks after surgery.

[0076] 3. Statistical analysis methods

[0077] All statistical analyses in this article were performed using GraphPad Prism 6.0 software. Data are expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test. Statistical significance was considered when *p < 0.05, **p < 0.01, or ***p < 0.001.

[0078] (2) Experimental results

[0079] 1. Effect of ZBP1 deficiency on the recovery of right forelimb motor function in mice after brachial plexus root avulsion and replantation surgery

[0080] A double-blind method was used to test the recovery of the mice's forelimb motor function using a face washing test every week after surgery. Figure 2 As shown in Figure B, the average score of mice in the ZBP1 knockout group was 4.14, with most (6 mice) ranging from 4 to 5 and only one mouse scoring 3. The average score of wild-type mice was 2.43, indicating that ZBP1 knockout can promote the recovery of motor function after brachial plexus root avulsion and replantation in mice.

[0081] 2. Effect of ZBP1 deficiency on the recovery of forelimb motor function in mice after brachial plexus root avulsion and replantation surgery

[0082] A double-blind method was used to test the recovery of the mice's forelimb motor function using the cylinder test every week after surgery. Figure 2 As shown in Figure C, in the first week, the ZBP1 knockout mice's right forepaw touched the cylinder wall an average of 7.4 times, while the WT mice's right forepaw touched the cylinder wall an average of only 3.6 times. By the eighth week, the ZBP1 knockout mice's right forepaw touched the cylinder wall an average of 14.6 times, while the WT mice's right forepaw touched the cylinder wall an average of 9.8 times. These experimental results show that in the first and eighth weeks, the ZBP1 knockout mice's right forepaw touched the cylinder wall significantly more often than the WT mice. This suggests that ZBP1 knockout can promote motor function recovery in mice after brachial plexus root avulsion and replantation.

[0083] Example 4

[0084] ZBP1 gene knockout reduces spinal cord anterior horn motor neuron death in a mouse model of brachial plexus root avulsion and reimplantation

[0085] 1. Animal model and grouping: Same as Example 1.

[0086] 2. Tissue preparation: as in Example 1.

[0087] 3. Neutral red staining

[0088] Prepare sections of frozen mouse spinal cord tissue and dry them at 37°C. Rinse in distilled water for several minutes to remove the OCT. Stain in neutral red stain for 10 minutes. Rinse in distilled water for several minutes. Pour sections into 95% and 100% ethanol for 5 minutes each. Pour sections into xylene I, xylene II, and xylene III for 5 minutes each. Mount the sections with neutral resin and oven-dry at 37°C overnight. Photograph, observe, and count the sections using an optical microscope.

[0089] 4. Real-time fluorescence quantitative PCR: The operation method is the same as Example 1.

[0090] Total RNA was extracted from mouse spinal cord tissue using TRIzol reagent. RNA was extracted from the tissue using the kit and reverse transcribed. mRNA expression levels of spinal cord apoptosis markers bax and caspase-3 were analyzed. Internal reference primers were the same as in Example 1.

[0091] Table 3 Real-time fluorescence quantitative PCR primer sequences

[0092]

[0093] 5. Statistical analysis methods

[0094] All statistical analyses in this article were performed using GraphPad Prism 6.0 software. Data are expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test. Statistical significance was considered when *p < 0.05, **p < 0.01, or ***p < 0.001.

[0095] (3) Experimental results

[0096] 1. ZBP1 gene knockout reduces spinal cord anterior horn motor neuron death in a mouse model of brachial plexus root avulsion and reimplantation

[0097] The results are as follows Figure 3 Figure A shows the number of motor neurons detected using neutral red staining. Compared with the WT group, the number of motor neurons in the ZBP1 knockout group was significantly increased. (A) Representative magnified image of the right anterior horn of the spinal cord stained with neutral red. Figure 3 B and 3C show that the expression levels of bax and caspase-3, which are related to spinal cord cell apoptosis, are decreased. The results suggest that ZBP1 gene knockout improves the survival of motor neurons in brachial plexus root avulsion and replantation and promotes motor function recovery.

[0098] Example 5

[0099] ZBP1 gene knockout inhibits pyroptosis and inflammation in spinal cord cells of mice with brachial plexus root avulsion and reimplantation

[0100] 1. Animal model and grouping: Same as Example 1.

[0101] 2. Tissue preparation: Same as Example 1.

[0102] 3. Real-time fluorescence quantitative PCR: the operation method is the same as Example 1.

[0103] Total RNA was extracted from mouse spinal cord tissue using TRIzol reagent. RNA was extracted from the tissue using the kit and reverse transcribed. mRNA expression levels of the apoptosis markers caspase-4, GSDMD, and NLRP3, and the inflammatory markers IL-6 and TNF-α were analyzed. Internal reference primers were the same as in Example 1.

[0104] Table 4 Real-time fluorescence quantitative PCR primer sequences

[0105]

[0106] 4. Statistical analysis methods

[0107] All statistical analyses in this article were performed using GraphPad Prism 6.0 software. Data are expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test. Statistical significance was considered when *p < 0.05, **p < 0.01, or ***p < 0.001.

[0108] (3) Experimental results

[0109] 1. ZBP1 gene knockout inhibits cell pyroptosis and inflammation

[0110] The results are as follows Figure 4 As shown in Figures A, 4B, 4C, 4D, and 4E, qRT-PCR results showed that compared with the WT group, the ZBP1 knockout group had decreased pyroptosis markers, including caspase-4, GSDMD, and NLRP3, and inflammatory markers, including IL-6 and TNF-α. These results suggest that ZBP1 knockout inhibits pyroptosis and inflammation in spinal cord cells in mice with brachial plexus root avulsion and reimplantation, promoting motor function recovery.

[0111] Example 6

[0112] ZBP1 gene knockout promotes remyelination of nerve fibers after brachial plexus root avulsion and replantation in mice

[0113] 1. Animal model and grouping: Same as Example 1.

[0114] 2. Tissue preparation: Same as Example 1.

[0115] 3. LFB staining

[0116] Take the frozen mouse nerve tissue sections that have been cut and dried at 37°C. Wash them in distilled water for several minutes to clean the OCT. First quickly rinse with 95% alcohol, then place them in 0.1% LFB staining solution in a 60°C oven for 16 hours. Rinse with distilled water for several minutes. Immerse them in 95% alcohol for a few seconds, separate them with 0.05% lithium carbonate aqueous solution for more than 10 seconds, and continue to differentiate them with 70% alcohol. Observe the staining effect under a microscope. Then, place them in 95% and 100% ethanol for 5 minutes each. Place them in xylene I, xylene II, and xylene III for 5 minutes each. Seal the slides with neutral resin and place them in a 37°C oven overnight. Take pictures, observe, and count them under an optical microscope.

[0117] 4. Tissue Immunofluorescence

[0118] Frozen mouse neural tissue sections were washed three times in PBS containing 0.1% Triton X-100. Sections were incubated with anti-choline acetyltransferase antibody at a 1:50 dilution overnight at 4°C, followed by incubation with fluorescent secondary antibody Alexa Fluor 594 at a 1:100 dilution for 1 hour at room temperature. Images were acquired using a confocal microscope with a 40× objective.

[0119] 5. Real-time fluorescence quantitative PCR: The operation method is the same as Example 1.

[0120] Total RNA was extracted from mouse spinal cord tissue using TRIzol reagent. RNA was extracted from the tissue using the kit and reverse transcribed. mRNA expression levels of the spinal cord myelination markers NGFR and Pou3f1 were analyzed.

[0121] Table 3 Real-time fluorescence quantitative PCR primer sequences

[0122]

[0123] The internal reference primers were the same as those in Example 1.

[0124] 6. Statistical analysis methods

[0125] All statistical analyses in this article were performed using GraphPad Prism 6.0 software. Data are expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test. Statistical significance was considered when *p < 0.05, **p < 0.01, or ***p < 0.001.

[0126] (3) Experimental results

[0127] 1. ZBP1 gene knockout improves the abnormal morphology of musculocutaneous nerve fibers in a model of brachial plexus root avulsion and replantation in mice

[0128] The results are as follows Figure 5 As shown in A, LFB staining revealed that the number of LFB-positive axons in the ZBP1 knockout group was significantly increased compared with the WT group. Figure 5 As shown in B, immunofluorescence staining results show that the number of Chat-positive motor neuron axons increases in ZBP1 knockout mice, indicating that ZBP1 knockout improves the abnormal morphology of musculocutaneous nerve fibers.

[0129] 2. ZBP1 gene knockout promotes remyelination of musculocutaneous nerve axons in BPRA mice

[0130] The results are as follows Figure 5 As shown in Figure C, qRT-PCR results showed that the expression of myelination markers ngfr and pou3f1 was reduced in the ZBP1 knockout group compared with the WT group, indicating that ZBP1 knockout reduced spinal cord myelination in mice with brachial plexus root avulsion and reimplantation.

[0131] The above results suggest that ZBP1 gene knockout improves the remyelination of motor nerve fibers in brachial plexus root avulsion and replantation, and promotes motor function recovery.

[0132] Example 7

[0133] ZBP1 gene knockout reduces muscle atrophy in mice following brachial plexus root avulsion and replantation

[0134] 1. Animal model and grouping: Same as Example 1.

[0135] 2. Tissue preparation: Same as Example 1.

[0136] 3. Biceps weighing

[0137] To study the degree of muscle atrophy after brachial plexus injury, the biceps brachii were weighed. The left and right biceps brachii were weighed using a weighing balance.

[0138] 4. H&E staining

[0139] Take the frozen mouse musculocutaneous nerve and biceps brachii tissue sections that have been cut and dried at 37°C. Wash in distilled water for several minutes to clean the OCT. Then place in hematoxylin staining solution for 10 minutes. Wash with distilled water and place in tap water. Then differentiate with 0.5% hydrochloric acid ethanol (prepared with 70% alcohol) for a few seconds and observe the effect under a microscope. Place in tap water and wash with distilled water. Place in eosin staining solution for 3 minutes. Wash with distilled water and observe the effect under a microscope. Place in 95% and 100% ethanol for 5 minutes each. Place in xylene I, xylene II, and xylene III for 5 minutes each. Seal the slides with neutral resin and place in a 37°C oven overnight. Take pictures, observe, and count with an optical microscope.

[0140] 5. Statistical analysis methods

[0141] All statistical analyses in this article were performed using GraphPad Prism 6.0 software. Data are expressed as mean ± standard deviation (SD) and analyzed using the Student's t-test. Statistical significance was considered when *p < 0.05, **p < 0.01, or ***p < 0.001.

[0142] (3) Experimental results

[0143] 1. ZBP1 gene knockout reduces muscle atrophy in mice with brachial plexus root avulsion and replantation

[0144] The effect of ZBP1 deficiency on muscle atrophy in mice after brachial plexus root avulsion and replantation surgery was analyzed by weighing and statistically analyzing the results. Figure 6 As shown in A, the muscle weight of mice in the ZBP1 knockout group was significantly greater than that of the wild-type group, indicating that the ZBP1 knockout group can alleviate biceps muscle atrophy after brachial plexus nerve root avulsion and replantation in mice.

[0145] 2. ZBP1 gene knockout reduces the number of fibroblast nuclei in mouse muscle

[0146] The results are as follows Figure 6 As shown in Figure B, H&E staining revealed an average of 59 fibroblast nuclei within randomly selected areas of muscle cells in the ZBP1 knockout group, compared to 17 in the WT group. This indicates that the number of fibroblast nuclei in ZBP1 knockout muscle was significantly reduced compared to the WT group. These experimental results indicate that ZBP1 knockout reduces the number of fibroblast nuclei in mouse muscle. These results suggest that ZBP1 knockout reduces muscle atrophy in mice following brachial plexus root avulsion and reimplantation.

[0147] It should be noted that the above embodiments are merely examples for the purpose of clearly illustrating the present invention and are not intended to limit the embodiments of the present invention. A person skilled in the art would be able to make other variations or modifications based on the above description. It is not possible to enumerate all embodiments here. Any obvious variations or modifications arising from the technical solution of the present invention remain within the scope of protection of the present invention.

Claims

1. Application of nucleic acid binding protein ZBP1 in the preparation of drugs for the treatment of brachial plexus root avulsion injury.

2. The use according to claim 1, characterized in that The application is the application of the nucleic acid binding protein ZBP1 in the preparation of a drug for promoting nerve regeneration and repair after brachial plexus root avulsion surgery.

3. The use according to claim 1, characterized in that Surgical treatment for brachial plexus root avulsions includes nerve root reimplantation.

4. Use of an agent for knocking out the ZBP1 gene or an agent for reducing the level of the ZBP1 gene in the preparation of a drug for treating brachial plexus root avulsion injury.

5. The use according to claim 4, characterized in that The reagents for knocking out the ZBP1 gene include CRISPR-Cas9, Cas9 nuclease that specifically cuts the ZBP1 gene, sgRNA that guides Cas9 to the ZBP1 gene, a transfection reagent containing the Cas9 nuclease that specifically cuts the ZBP1 gene and the sgRNA that guides Cas9 to the ZBP1 gene, TALEN protein targeting the ZBP1 gene, a vector or transfection reagent containing the TALEN protein targeting the ZBP1 gene, siRNA that can specifically bind to and degrade the ZBP1 gene mRNA, and a transfection reagent containing siRNA that specifically binds to and degrades the ZBP1 gene mRNA.

6. The use according to claim 4, characterized in that The application is the use of a reagent for knocking out the ZBP1 gene or a reagent for reducing the level of the ZBP1 gene in the preparation of a drug for promoting nerve regeneration and repair after brachial plexus root avulsion surgery.

7. The use according to claim 4, characterized in that The reagents for reducing the level of the ZBP1 gene include: siRNA that can complementarily pair with a specific sequence of the ZBP1 gene mRNA, a transfection reagent containing siRNA that can complementarily pair with a specific sequence of the ZBP1 gene mRNA, a kit containing siRNA that can complementarily pair with a specific sequence of the ZBP1 gene mRNA, miRNA targeting ZBP1 mRNA, a short single-stranded DNA or RNA molecule complementary to ZBP1 mRNA, inactivated Cas9 that binds to the promoter or enhancer region of the ZBP1 gene, Cas9 nuclease that specifically cuts the ZBP1 gene, sgRNA that guides Cas9 to the ZBP1 gene, a transfection reagent containing Cas9 nuclease that specifically cuts the ZBP1 gene and sgRNA that guides Cas9 to the ZBP1 gene, TALEN protein targeting the ZBP1 gene, a vector or transfection reagent containing TALEN protein targeting the ZBP1 gene, siRNA that can specifically bind to and degrade the ZBP1 gene mRNA, and a transfection reagent containing siRNA that specifically binds to and degrades the ZBP1 gene mRNA.

8. The use according to any one of claims 1 to 7, characterized in that The drug further comprises one or more pharmaceutically acceptable carriers, including coating materials, solvents, solubilizers, adhesives, stabilizers, antioxidants, pH regulators, and flavoring agents.

9. The use according to any one of claims 1 to 7, characterized in that: The dosage form of the medicine includes any one of tablets, capsules, granules, powders, powder injections, pills, emulsions, suspensions, and solutions.

10. The use according to any one of claims 1 to 7, characterized in that The administration of the drug includes any one of injection, oral administration, absorption, physical or chemical mediation to introduce the drug into the body; or the drug can be mixed or wrapped with other substances and then introduced into the body.

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