The use and intervention methods of Chek2 inhibitors in the preparation of drugs for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders.

Abstract

This invention discloses the use of Chek2 inhibitors in the preparation of drugs for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders, and its intervention methods, relating to the field of biomedical technology. This invention provides the use of Chek2 inhibitors in the preparation of drugs for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders. This invention is the first to discover and validate Chek2 as a key molecular target linking "chronic sleep disorders" and "postoperative pain sensitization." By constructing a composite animal model of chronic sleep deprivation (CSD) combined with postoperative incision pain, the pathological process of increased postoperative pain in patients with sleep disorders was simulated. Using mRNA-seq and Western blot techniques, this invention is the first to discover and confirm that the expression level of Chek2 in the nucleus accumbens (NAc) brain region is significantly upregulated during postoperative pain sensitization induced by chronic sleep disorders, filling a gap in the field of "sleep disorder-pain interaction" regarding specific central targets.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically the use of Chek2 inhibitors in the preparation of drugs for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders, and the intervention method thereof. Background Technology

[0002] Postoperative pain management is one of the core issues in perioperative medicine. Despite continuous advancements in analgesia techniques, a significant proportion of patients still experience poor acute pain control or chronic pain postoperatively, a condition known as postoperative hyperalgesia. This hyperalgesia manifests not only as a significant decrease in mechanical and thermal pain thresholds but may also lead to increased spontaneous pain, severely impacting patients' postoperative functional exercises, quality of life, and long-term rehabilitation outcomes.

[0003] Clinically, the number of patients with pre-existing chronic sleep disturbances (CSD) is growing rapidly. Epidemiological evidence suggests that chronic sleep deprivation or sleep fragmentation is an independent risk factor for increased postoperative pain and the development of chronic pain. Compared to patients with normal sleep, those with sleep disturbances often report higher pain scores, require more opioids, experience longer durations of pain, and have a significantly increased risk of developing chronic postoperative pain under the same surgical trauma. However, current interventions for this specific clinical phenomenon are extremely limited. Existing analgesic regimens primarily target peripheral inflammation or spinal cord nociceptive signaling, failing to address the core pathological mechanism of the central facilitatorship process of "chronic sleep disturbance → postoperative pain sensitization." The key reason for this lies in the lack of in-depth understanding of the common central targets connecting sleep disturbances and pain regulation, as well as their druggable pathways.

[0004] The brain's reward and motivation circuitry plays a crucial role in the cognitive-emotional dimension of pain perception. The nucleus accumbens (NAc), as the hub of this circuitry, receives extensive input from the prefrontal cortex, hippocampus, amygdala, and ventral tegmental area, integrating stress, emotion, reward anticipation, and sensory information. Recent studies suggest that NAc dysfunction is closely related to emotional disturbances and pain maintenance in chronic pain states, and that sleep deprivation can significantly alter NAc neuronal activity and synaptic plasticity. However, the specific molecular changes occurring within the NAc brain region in the context of postoperative pain sensitization induced by chronic sleep disorders, and the existence of potentially modifiable kinase targets, have not been systematically reported previously.

[0005] Checkpoint kinase 2 (Chek2) has traditionally been considered a key kinase in DNA damage response and cell cycle regulation. However, its function in the mature central nervous system remains largely unexplored. Recent studies suggest that Chek2 may be involved in the regulation of neuronal responses to stress stimuli and synaptic remodeling. As a stress-related kinase, the role and interventional value of Chek2 in postoperative pain sensitization induced by chronic myocardial infarction (CSD) have not yet been systematically proposed and validated.

[0006] In summary, current technologies lack specific central nervous system-targeted intervention programs for chronic sleep disorders that induce or exacerbate postoperative pain sensitization. Based on novel mechanistic findings, this invention proposes a new use for Chek2 inhibitors in this indication and specific intervention methods, providing a solid scientific basis and a potentially efficient translational strategy for addressing the aforementioned clinical challenges.

[0007] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0008] The purpose of this invention is to provide the use of Chek2 inhibitors in the preparation of medicaments for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders, and methods of intervention thereof, in order to solve the problems in the prior art.

[0009] This invention employs a chronic sleep deprivation protocol to induce more severe and persistent pain sensitization based on a postoperative incision pain model, accompanied by a neural structural plasticity phenotype (increased dendritic spine density). mRNA-seq differential analysis showed significant upregulation of Chek2; Western blotting confirmed elevated Chek2 protein levels. Chek2 was knocked down by NAc viral injection, reversing pain sensitization and correcting the synaptic plasticity phenotype. The Chek2 small molecule inhibitor CCT241533 was administered, with a dose gradient established to screen for the optimal dose and demonstrate its ability to improve pain sensitization.

[0010] To achieve the above objectives, the present invention provides the following technical solution: the use of a Chek2 inhibitor in the preparation of a medicament for the prevention and / or treatment of postoperative pain sensitization induced or aggravated by chronic sleep disorders, wherein the Chek2 inhibitor exerts its effect by inhibiting Chek2 activity and / or reducing Chek2 expression.

[0011] Preferably, the chronic sleep disorder is a sleep disorder caused by chronic sleep deprivation or sleep fragmentation.

[0012] Preferably, the postoperative pain sensitization includes at least one of the following: mechanical sensitization, thermal sensitization, or increased spontaneous pain.

[0013] Preferably, the Chek2 inhibitor is selected from: small molecule inhibitors, siRNA, shRNA, antisense oligonucleotides, CRISPRi system, or combinations thereof.

[0014] A method for intervening in postoperative pain sensitization induced or aggravated by chronic sleep disorders, the method being a non-therapeutic experimental research method, comprising the step of administering an effective amount of Chek2 inhibitor to the subject; the intervention method improves postoperative pain sensitization behavior induced by chronic sleep disorders and / or reduces the density of dendritic spines of neurons in the nucleus accumbens brain region by inhibiting Chek2 activity and / or reducing Chek2 expression levels.

[0015] The Chek2 inhibitor was administered to the nucleus accumbens (NAc) brain region via stereotactic injection.

[0016] Preferably, the Chek2 inhibitor is a Chek2-targeting shRNA viral vector, and the coordinates of the shRNA viral vector injected into the nucleus accumbens brain region are: with the anterior fontanelle (Bregma) as the reference point, AP +1.50 mm, ML ±1.00 mm, DV −4.40 mm.

[0017] Preferably, the injection volume of the shRNA viral vector is 1.0 μL per side of the nucleus accumbens, the injection rate is 100 nL / min, and the needle is left in place for 10-15 minutes after injection.

[0018] Preferably, the Chek2 inhibitor is administered systemically via intraperitoneal injection (ip).

[0019] Preferably, the Chek2 inhibitor is the small molecule compound CCT241533, with an effective intervention dose of 1 mg / kg. The administration regimen of CCT241533 is once daily, continuously administered during the postoperative period.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. This invention is the first to discover and validate Chek2 as a key molecular target linking "chronic sleep disorders" and "postoperative pain sensitization." By constructing a composite animal model of chronic sleep deprivation (CSD) combined with postoperative incision pain, the pathological process of increased postoperative pain in patients with sleep disorders was simulated. Using mRNA-seq and Western blot techniques, the expression level of Chek2 in the nucleus accumbens (NAc) brain region was significantly upregulated during postoperative pain sensitization induced by chronic sleep disorders, filling a gap in the field of "sleep disorder-pain interaction" regarding specific central targets. This provides a novel theoretical basis and target for developing precision intervention drugs targeting this specific patient population.

[0022] 2. This invention elucidates the specific function of Chek2 in the nucleus accumbens (NAc) and its downstream neural plasticity mechanism. It confirms that chronic sleep disorders can lead to an abnormally increased density of dendritic spines in neurons within the NAc, a change in synaptic structural plasticity closely related to chronic pain. By targeting and knocking down Chek2 expression within the NAc, not only can pain sensitization be effectively alleviated, but this abnormal neural structural remodeling can also be significantly reversed. This reveals a novel mechanism by which Chek2 mediates pain sensitization by regulating synaptic plasticity in the central reward / motivation circuit, directly linking the molecular level of intervention to changes in neural circuit structure, thus deepening our understanding of the central pain regulation network.

[0023] 3. This invention provides several practical and efficient Chek2 intervention strategies, including gene silencing and pharmacological inhibition, with extremely high clinical translational potential. This invention provides a Chek2-targeting shRNA viral vector and its precise stereotactic injection parameters into the nucleus accumbens. In animal experiments, this method has been shown to efficiently and specifically silence the target gene, effectively reversing pain sensitization, providing precise experimental evidence and operational standards for gene therapy strategies. This invention screened and validated the efficacy and optimal dosage of the small molecule inhibitor CCT241533. CCT241533 demonstrated good efficacy in improving postoperative pain sensitization, and compared to gene therapy, it has significant advantages in terms of more convenient administration and higher clinical feasibility. This provides candidate compounds and dosing regimens for the development of novel oral or injectable analgesics.

[0024] 4. This invention not only systematically elucidates the core role and mechanism of Chek2 in postoperative pain sensitization induced by chronic sleep disorders from multiple levels, including molecular, cellular, circuit, and behavioral aspects, but also provides multi-dimensional intervention programs with high translational potential, including gene silencing and small molecule inhibitors. It has important scientific value and broad clinical application prospects for improving pain management in patients with perioperative sleep disorders, reducing the incidence of postoperative chronic pain, and accelerating patient recovery. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0026] Figure 1 Timeline of CSD+incision model (days 1-7 8:00-14:00 6h SD daily; day 8 right plantar incision; day 9-18 continued 6h SD and von Frey) (A), and CSD-surgery group showed a more significant decrease in PWT (B) and increased dendritic spine density (C).

[0027] Figure 2 CSD vs control differential analysis volcano plot / heatmap showed Chek2 upregulation (AB); NAc WB verified the Chek2 protein change (C);

[0028] Figure 3 : NAc injection localization and fluorescence expression (A); Chek2-shRNA improves PWT (B) and reduces dendritic spine density (C);

[0029] Figure 4 CCT241533 ip dosing timeline (A); the optimal dose gradient (0 / 0.3 / 1 / 3 mg / kg) was selected to be 1 mg / kg (B); and the changes in Chek2 expression level in the NAc brain region after 1 mg / kg dosing are shown (C). Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Example 1: Establishing a chronic sleep disorder (CSD) combined with right foot incision pain model, and detecting changes in pain threshold behavior and NAc synaptic plasticity in mice.

[0032] 1. Laboratory animals

[0033] Male C57BL / 6J mice aged 8-10 weeks were selected and purchased from Vital River Pharmaceuticals in Beijing. Five mice were housed per cage at a temperature of 22±2℃ and a humidity of 55±10%. Food and water were provided freely. A 12-hour light-dark cycle (8:00-20:00 light) was established before sleep deprivation. All experimental procedures strictly adhered to animal ethics guidelines and were approved by the Animal Ethics Committee of Wenzhou Medical University.

[0034] 2. Methods

[0035] 2.1 CSD Model Establishment (by Figure 1 (Technical route)

[0036] After 1 week of acclimatization (establishing a light-dark cycle), mice were subjected to chronic sleep deprivation (CSD): 6 hours of sleep deprivation were performed daily from 8:00 to 14:00 for 7 days before surgery and 9 days after surgery; the rest of the time, the mice were returned to their regular cages. The sleep deprivation method was a modified water platform method, in which the animals had to remain alert to avoid falling into the water and could not fall asleep, thus causing sleep deprivation. The control group mice were placed in the same environment but their sleep was not restricted (waterless control).

[0037] 2.2 Establishment of the right foot plantar incision model

[0038] After 7 days of sleep deprivation before surgery, mice were anesthetized with 1.5% isoflurane to establish a right foot incision pain model. After shaving and disinfection, a standardized incision (5-7 mm in length) was made on the right hind foot, cutting through the skin and superficial tissues to avoid damage to the major blood vessels and nerves of the foot. The incision was then sutured, and routine postoperative care was provided and recovery was observed.

[0039] 2.3 Mechanical pain threshold (von Frey) behavioral test

[0040] Before testing, mice were placed in a quiet, temperature-controlled (22-25℃) behavioral chamber for at least 1 hour and acclimatized in a transparent test cage with a metal mesh bottom for 30 minutes. The right hind paw was stimulated with standard Von Frey fibers, the fibers perpendicularly contacting the center of the paw and bending into a "C" shape for 3-6 seconds. A positive response was recorded if the mouse exhibited rapid paw withdrawal, shaking, licking, or sustained paw lifting after stimulation. The 50% mechanical withdrawal threshold (PWT) was calculated using the up-and-down method. The same limb was repeated 2-3 times, and the average value was taken.

[0041] 2.4 Sparse Marking and Dendritic Thorn Density Detection

[0042] 1) Sparsely labeled virus injection (NAc)

[0043] Sparsely labeled virus injection method: After anesthesia, mice were fixed in a stereotaxic apparatus, and the NAc coordinates (AP +1.50, ML ±1.00, DV −4.40 mm) were determined according to brain atlas. NCSP-RFP-2E5 sparsely labeled virus was slowly injected using a microinfusion pump at a rate of 50 nL / min, with an injection volume of 300 nL per side. The needle was left in place for 10-15 minutes after injection to reduce backflow. After injection, mice were kept in standard feeding conditions for 14 days to ensure stable expression of the fluorescent protein in neurons and sufficient filling of dendritic structures.

[0044] 2) Model processing and material acquisition time points

[0045] After waiting 14 days for viral expression, a chronic sleep deprivation combined with incision pain model was routinely established. Based on the fact that the pain threshold of the behavioral control group returned to normal level on the ninth day after surgery, samples were taken on the ninth day after surgery to analyze the changes in dendritic spine density of neurons in the NAc brain region when chronic sleep deprivation caused prolonged pain.

[0046] 3) Cardiac perfusion and tissue fixation

[0047] Mice were deeply anesthetized and their thoracic cavity was exposed. A perfusion needle was inserted into the left ventricle, and the right atrium was cut open to establish an outflow pathway. The effluent was first perfused with pre-cooled saline or PBS until it was clear and free of obvious blood color to reduce the influence of residual hemoglobin on fluorescence observation. Then, the effluent was fixed and perfused with 4% paraformaldehyde (PFA). After the brain tissue was removed, it was placed in fresh 4% PFA for further fixation for 24 hours. Gradient glucose immersion was then performed (10%→20%→30%, each step lasting approximately 24 hours). Serial coronal sections with a thickness of 40 µm were cut using a cryostat.

[0048] 4) Slice preparation and fluorescence imaging processing

[0049] NAc brain sections were gently rinsed with PBS to remove residual fixative / sucrose, attached to slides, and then covered with mounting medium containing anti-quenching agents. The slides were sealed with coverslips and stored in the dark. Dendritic spine images were acquired using a Zeiss 980 high-resolution confocal microscope under a 63× oil immersion lens. Multiple neurons with clear, sparsely labeled dendrites, low background, and intact dendrites were selected from each animal. To minimize interference from inherent differences in different dendritic segments, dendritic segments far from the cell body but still stably recognizable as spines were prioritized for statistical analysis. Dendritic spines were identified and counted using microscope software or image analysis software. The calculation formula is as follows:

[0050] Dendritic spine density = Number of dendritic spines / Dendritic length (spines / µm)

[0051] 3. Results of this embodiment

[0052] 1) Compared with the surgery group, the CSD-surgery group showed a significantly lower mechanical withdrawal threshold from day 1 to day 9 postoperatively (P<0.01), and the pain threshold recovery time was significantly delayed until after day 9 postoperatively, suggesting that chronic sleep disorder can induce / exacerbate postoperative pain sensitization. Figure 1 B).

[0053] 2) Morphological results showed that the spinal dynamite density of NAc neurons was significantly increased in the CSD-surgery group ( Figure 1 (C) suggests that the model is accompanied by enhanced synaptic plasticity, indicating that chronic sleep disorders induce synaptic remodeling, which is closely related to postoperative persistent pain sensitization, providing a pathological basis for subsequent molecular mechanisms and interventions.

[0054] Example 2: mRNA sequencing screening and verification of Chek2 upregulation in CSD-induced postoperative pain sensitization

[0055] 1. Method

[0056] 2.1 NAc tissue sampling

[0057] Mice were sacrificed on the 9th day after surgery, and NAc tissue was rapidly isolated according to mouse brain atlas, flash-frozen in liquid nitrogen, and used for mRNA sequencing and protein verification.

[0058] 2.2 mRNA sequencing and bioinformatics analysis

[0059] RNA extraction: Total RNA was extracted with Trizol, and its concentration and purity were detected by Nanodrop (A260 / A280=1.8-2.1). RIN was evaluated with Agilent 2100 (>7). rRNA was removed to enrich mRNA, which was then fragmented, reverse transcribed to synthesize cDNA, and a library was constructed. The mRNA was then sequenced using Illumina.

[0060] Sequencing data processing: FastQC / MultiQC quality control, trimmomatic adapter removal and low-quality data removal, HISAT2 alignment to mouse GRCm39 reference genome, featureCounts counting, DESeq2 differential analysis. Differential screening criterion: pvalue <0.05.

[0061] 2.3 Western blot validation of Chek2 protein expression

[0062] NAc tissue was lysed with RIPA containing a protease inhibitor, centrifuged, and the supernatant was collected. BCA quantification was performed. 30 μg of sample was loaded into each well. After SDS-PAGE electrophoresis, the sample was transferred to a PVDF membrane. Blocking was performed with rapid blocking buffer for 1 hour, followed by incubation overnight at 4°C with Chek2 primary antibody (1:1000). Secondary antibody (1:10000) was used at room temperature for 1 hour, followed by washing three times with TBST for 10 minutes each time, and ECL development. β-actin was used as an internal control. ImageJ was used to quantify the grayscale ratio (…). Figure 2 C).

[0063] 3. Results of this embodiment

[0064] 1) Differential analysis of mRNA sequencing showed that in hyperalgesic mice that underwent chronic sleep deprivation surgery, the transcriptional level of Chek2 was significantly upregulated. Figure 2 AB).

[0065] 2) Western blot results showed that Chek2 protein expression was significantly increased in NAc ( Figure 2 C), consistent with the transcriptome, suggests that Chek2 may be a key molecular target for CSD-induced postoperative pain sensitization.

[0066] Example 3: NAc knockdown of Chek2 and intervention with the Chek2 small molecule inhibitor CCT241533 to evaluate its effect on improving postoperative pain sensitization and spinal process density induced by CSD. This example consists of two parts: (A) gene knockdown verification and (B) pharmacological verification and Chek2 small molecule inhibitor verification.

[0067] 2. Methods

[0068] The effect of A. NAc brain region knockdown of Chek2 expression on pain threshold

[0069] 2.1 NAc stereotactic injection coordinates: Using Bregma as the reference point, perform bilateral NAc injections (AP +1.50 mm, ML ±1.00 mm, DV −4.40 mm). Injection volume: 1.0 μL / side, injection speed: 100 nL / min. After injection, leave the needle in place for 10-15 minutes to prevent backflow and wait for the virus expression to complete.

[0070] 2.2 Modeling and Behavioral Analysis: After viral expression was completed, a CSD+ right foot incision model was established according to Example 1, and von Frey detection was performed continuously after the operation.

[0071] 2.3 Dendritic spine density detection

[0072] Same as 2.4 of Example 1.

[0073] The effect of B.CCT241533 (a small molecule inhibitor of Chek2) pharmacological intervention on pain threshold

[0074] 2.4 Dosing regimen

[0075] In the pharmacological intervention experiment, the Chek2 small molecule inhibitor CCT241533 was administered via intraperitoneal injection (ip). To determine the effective dose range, a dose gradient of 0, 0.3, 1, and 3 mg / kg was initially established, and the drug was administered once daily during the postoperative phase. After screening based on behavioral results, 1 mg / kg was determined as the working dose for subsequent validation (showing the best improvement trend, while the effects of 3 mg / kg and 1 mg / kg were similar). The dosing time window was consistent with the intervention window for continued CSD after surgery, and von Frey mechanical pain threshold testing was performed concurrently during the dosing period to assess the analgesic / anti-sensitization effect.

[0076] 2.5 Western blot validation of Chek2 protein expression

[0077] After injecting CCT241533 into the NAc brain region, Chek2 protein expression was detected, following the same procedure as in Example 1, section 2.3.

[0078] 3. Results of this embodiment

[0079] Part A (NAc knockdown): Compared with the scramble control group, NAc knockdown of Chek2 significantly increased the mechanical pain threshold and shortened the duration of pain sensitization in CSD-surgery mice. Figure 3 A); it can also significantly reduce the phenotype of increased NAc ridge density (A); Figure 3 B), suggesting that Chek2 has a causal role in pain sensitization within NAc.

[0080] Part B (Pharmacological Intervention): Intraperitoneal injection of CCT241533 improved pain sensitization induced by CSD-surgery. Dosage gradient results showed that the 1 mg / kg group exhibited the most significant improvement, thus determining it as the preferred dose. Furthermore, the daily postoperative dosing regimen steadily increased the mechanical pain threshold. Figure 4 B). Western blotting results support the possibility that this intervention can regulate Chek2-related protein levels (B). Figure 4 C).

[0081] This invention reveals for the first time that the upregulation of Chek2 in the nucleus accumbens (NAc) is a key molecular event linking chronic sleep disorders and postoperative analgesia. Stereotactic injection of Chek2-shRNA to knock down expression, or intraperitoneal injection of the Chek2 small molecule inhibitor CCT241533, effectively corrected abnormal synaptic plasticity in the NAc brain region, significantly alleviating and reversing persistent postoperative analgesia induced by chronic sleep disorders. This invention provides a novel intervention target and drug candidate for pain management in patients with perioperative sleep disorders.

[0082] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. The use of Chek2 inhibitors in the preparation of medicaments for the prevention and / or treatment of postoperative pain sensitization induced or exacerbated by chronic sleep disorders, characterized in that: The Chek2 inhibitor works by inhibiting Chek2 activity and / or reducing Chek2 expression.

2. The use according to claim 1, characterized in that: The chronic sleep disorder is a sleep disorder caused by chronic sleep deprivation or sleep fragmentation.

3. The use according to claim 1, characterized in that: The postoperative pain sensitization includes at least one of the following: mechanical sensitization, thermal sensitization, or increased spontaneous pain.

4. The use according to claim 1, characterized in that: The Chek2 inhibitor is selected from: small molecule inhibitors, siRNA, shRNA, antisense oligonucleotides, CRISPRi system or combinations thereof.

5. A method for intervening in postoperative pain sensitization induced or aggravated by chronic sleep disorders, characterized in that, The method is a non-therapeutic experimental research method, which includes the step of administering an effective amount of Chek2 inhibitor to the subject; the intervention method improves postoperative pain sensitization behavior induced by chronic sleep disorder and / or reduces the density of dendritic spines of neurons in the nucleus accumbens brain region by inhibiting Chek2 activity and / or reducing Chek2 expression levels.

6. The method according to claim 5, characterized in that: The Chek2 inhibitor is administered to the nucleus accumbens (NAc) brain region via stereotactic injection.

7. The method according to claim 6, characterized in that: The Chek2 inhibitor is a Chek2-targeting shRNA viral vector. The coordinates of the shRNA viral vector injected into the nucleus accumbens brain region are: with the anterior fontanelle (Bregma) as the reference point, AP +1.50 mm, ML ±1.00 mm, DV −4.40 mm.

8. The method according to claim 7, characterized in that: The injection volume of the shRNA viral vector is 1.0 μL per side of the nucleus accumbens, the injection rate is 100 nL / min, and the needle is left in place for 10-15 minutes after injection.

9. The method according to claim 5, characterized in that: The Chek2 inhibitor is administered systemically via intraperitoneal injection (ip).

10. The method according to claim 9, characterized in that: The Chek2 inhibitor is a small molecule compound, CCT241533, with an effective intervention dose of 1 mg / kg. The CCT241533 is administered once daily, continuously during the postoperative period.

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