Use of agents targeting camk2d in the preparation of a medicament for improving or treating postoperative cognitive dysfunction

Abstract

The application provides application of a reagent targeting CaMK2delta in preparation of a drug for improving or treating postoperative cognitive dysfunction, and belongs to the technical field of drug preparation. The application provides application of the reagent targeting CaMK2delta in preparation of the drug for improving or treating postoperative cognitive dysfunction. CaMK2delta is significantly and continuously up-regulated in an acute cognitive impairment period, and inhibition of CaMK2delta expression can realize improvement and treatment of postoperative cognitive dysfunction.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparation technology, specifically relating to the application of reagents targeting CaMK2δ in the preparation of drugs to improve or treat postoperative cognitive dysfunction. Background Technology

[0002] In recent years, the proportion of elderly patients requiring general anesthesia and major surgical procedures has been increasing year by year. Against this backdrop, postoperative cognitive dysfunction (POCD), as a common central nervous system complication, has attracted widespread attention in the fields of clinical anesthesiology and neuroscience. Clinically, POCD is mainly characterized by postoperative difficulty concentrating, decreased executive function, and progressive decline in spatial learning and memory. This cognitive impairment not only severely hinders accelerated postoperative recovery after surgery (ERAS) in elderly patients, significantly prolongs hospital stays, and increases the consumption of medical resources, but also has a profound impact on patients' long-term quality of life. However, due to the highly complex pathophysiological mechanisms of POCD, involving the trans-tissue transmission of peripheral systemic stress to the central nervous system, targeted prevention strategies and effective therapeutic drugs are still lacking in clinical practice. Therefore, in-depth analysis of the pathogenesis of POCD at the molecular level, and on this basis, identification of core target genes that can directly improve or treat the condition, is a core scientific problem that urgently needs to be solved in perioperative neuroscience. Summary of the Invention

[0003] The purpose of this invention is to provide the application of agents targeting CaMK2δ in the preparation of drugs for improving or treating postoperative cognitive impairment. CaMK2δ is significantly and persistently upregulated during the acute cognitive impairment period. By targeting CaMK2δ, inhibiting its expression can improve and treat postoperative cognitive impairment.

[0004] This invention provides the application of a CaMK2δ-targeting agent in the preparation of a drug for improving or treating postoperative cognitive dysfunction; the CaMK2δ-targeting agent is a CaMK2δ-inhibiting agent.

[0005] Preferably, the reagent for inhibiting CaMK2δ includes reagents that inhibit CaMK2δ activity, reagents that inhibit CaMK2δ gene transcription, or reagents that inhibit CaMK2δ protein expression.

[0006] The present invention also provides the application of CaMK2-targeting reagents in the preparation of drugs for improving or treating postoperative cognitive dysfunction; wherein the CaMK2-targeting reagent is a reagent that inhibits CaMK2.

[0007] Preferably, the reagent for inhibiting CaMK2 includes reagents that inhibit CaMK2 activity, reagents that inhibit CaMK2 gene transcription, or reagents that inhibit CaMK2 protein expression.

[0008] Preferably, the CaMK2 includes CaMK2α, CaMK2β, CaMK2γ, or CaMK2δ.

[0009] The present invention also provides the use of KN-93 in the preparation of medicaments for improving or treating postoperative cognitive impairment.

[0010] This invention also provides the application of reagents for detecting CaMK2 expression levels in the preparation of kits for diagnosing postoperative cognitive impairment.

[0011] This invention provides the application of agents targeting CaMK2δ in the preparation of drugs to improve or treat postoperative cognitive impairment. CaMK2δ is significantly and persistently upregulated during the acute cognitive impairment phase. Using it as a target gene, inhibiting CaMK2δ expression can improve and treat postoperative cognitive impairment. Experimental results show that CaMK2δ is a key candidate pathogenic gene driving the development of POCD, and inhibiting CaMK2δ activity can effectively alleviate impairment in spatial learning, reference memory, and working memory in POCD rats. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0013] Figure 1 The following diagrams illustrate the key time points revealing cognitive impairment in POCD rats using a combined Morris water maze and Y-maze test, provided by this invention. A is a simplified flowchart of the experiment; B is a schematic diagram of the escape latency in the water maze during the training period; C is a diagram showing the escape latency results; D is a swimming speed diagram; E is a representative image of the escape latency for each group during the experimental period; F is a diagram showing the time spent in the target quadrant; G is a diagram showing the number of times the platform was crossed; H is a diagram showing the escape latency results; I is a diagram showing the spontaneous alternation rate in the Y-maze; and J is a representative image of the spontaneous alternation rate in the Y-maze. Figure 2 The results of pseudo-temporal analysis and experimental verification provided by this invention identify Camk2d as the core candidate gene mediating POCD are shown in the figure below; where A is the pseudo-temporal analysis result figure; B is the KEGG pathway enrichment analysis figure; C is the expression heatmap of 8 key genes involved in the calcium signaling pathway; and D is the fluorescence quantitative result figure. Figure 3 The images provided by this invention show the results of significantly reversing postoperative cognitive impairment in aged rats by inhibiting Camk2δ; where A is a representative image of escape latency during the training period; B is a result of the decreasing trend of latency; C is a representative image of escape latency during the experimental period; D is a result of swimming speed; E is a result of time spent in the target quadrant; F is a result of the number of crossings; G is a result of escape latency; H is a result of the spontaneous alternation rate of the Y maze; and I is a representative image of the spontaneous alternation rate of the Y maze. Detailed Implementation

[0014]

[0015] This invention also provides the application of CaMK2-targeting agents in the preparation of drugs for improving or treating postoperative cognitive impairment; the CaMK2-targeting agents are CaMK2 inhibitors. KN-93, as a CaMK2 inhibitor, can improve and treat POCD. Examples demonstrate that inhibiting the expression of CaMK2 family members can improve and treat POCD. In specific embodiments, the CaMK2-inhibiting agents include agents that inhibit CaMK2 activity, agents that inhibit CaMK2 gene transcription, or agents that inhibit CaMK2 protein expression.

[0016] This invention also provides the application of KN-93 in the preparation of medicaments for improving or treating postoperative cognitive impairment. Experimental results show that KN-93 can achieve improvement and treatment of POCD.

[0017] This invention also provides the application of reagents for detecting CaMK2 expression levels in the preparation of kits for diagnosing postoperative cognitive impairment. CaMK2δ is a key candidate pathogenic gene driving the development of POCD, and detecting CaMK2 expression levels can aid in the diagnosis of postoperative cognitive impairment.

[0018] To further illustrate the present invention, the application of the CaMK2δ inhibitor provided by the present invention in the preparation of medicaments for improving or treating postoperative cognitive dysfunction is described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1 Laboratory animals and grouping: The experiment was conducted in two phases: In the molecular screening phase, rats were randomly divided into 4 groups (n=8): normal control group (Ctrl), postoperative day 3 group (POD3), postoperative day 5 group (POD5), postoperative day 7 group (POD7); In the drug intervention phase, rats were randomly divided into 3 groups (n=8): normal control group (Ctrl), POCD model group (POCD), and POCD+KN-93 administration group (POCD+KN-93).

[0020] The combined Morris water maze and Y-maze test revealed that the key time point for cognitive impairment in POCD rats was day 3 post-surgery. The apparatus consisted of a circular black pool, 150 cm in diameter and 60 cm high, filled with water at a suitable temperature, ensuring the rats' heads remained above water. The pool was divided into four quadrants. For five consecutive days prior to surgery, four groups of aged rats from the molecular screening phase underwent water maze training (four times a day, 60 seconds each time), recording their escape latency to assess their learning and memory abilities. After the training period, postoperative spatial exploration was conducted, recording the escape latency, target quadrant dwell time, and platform crossings for each group within 60 seconds. All data were automatically analyzed using Tracking Master V5.0 software. A standard Y-maze was used for spontaneous alternation experiments, with the three arms forming 120° intervals, labeled A, B, and C. The target arm was not fixed during the experiment. In the formal experiment, the rats were placed in the center of the maze and allowed free exploration for 10 minutes each time to adapt to the environment. The total number of arm entries and the effective spontaneous alternation rate were extracted during the formal experiment. After the behavioral tests of arms A, B, and C, the rats in each group were euthanized at the corresponding time points, and hippocampal tissue was rapidly separated for subsequent tissue collection.

[0021] This invention uses splenectomy to construct a postoperative cognitive impairment model in aged rats. Figure 1 In the diagram, A represents a simplified flowchart of the experiment. Figure 1 B in the diagram represents the escape latency of the Morris water maze during the training period. The results of the Morris water maze training period showed no significant differences in the decreasing trend of latency and swimming speed (approximately 30 cm / s) among the groups, with consistent baselines. This indicates that there were no differences in motor ability and learning / memory ability among the groups before surgery. Figure 1 C and Figure 1 Figure D shows that postoperative behavioral tests consistently indicated significant cognitive impairment in rats, most severe on postoperative day 3, followed by gradual recovery over time. Figure E shows representative images of escape latency for each group during the experimental period. Compared to the control group, the surgical group had the shortest time spent in the target quadrant of the Morris water maze on postoperative day 3 (approximately 10 seconds). Figure 1 (F) has the fewest platform crossings (approximately 1 time). Figure 1 (G) has the longest incubation period (approximately 40 seconds). Figure 1 In the H section, the spontaneous alternation rate of the Y maze also drops to its lowest point (approximately 55%). Figure 1 I and Figure 1 (J in the original text) Although the above indicators rebounded at subsequent time points, they did not fully recover. In summary, the third day after surgery is a critical time point for the occurrence of postoperative cognitive dysfunction in aged rats.

[0022] Pseudo-temporal analysis and experimental validation identified Camk2d as the core candidate gene mediating POCD: Real-time fluorescence reverse transcription quantitative polymerase chain reaction (RT-qPCR): Total RNA was extracted from hippocampal tissue using TRIzol reagent (Invitrogen, USA), and RNA concentration and purity were detected using a NanoDrop spectrophotometer (Thermo Fisher, USA). 1 μg of total RNA was used to synthesize cDNA using the PrimeScript reverse transcription kit (Takara, Japan). Amplification was performed using PerfectStart Green qPCR premix (TransGen Biotech, Beijing) on ​​a CFX96 real-time quantitative PCR instrument (Bio-Rad) under the following conditions: 95℃ for 30 s; 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control, and the relative mRNA expression level of the target gene was calculated using the 2^(-ΔΔCt) method.

[0023] Transcriptome sequencing and bioinformatics analysis: Total RNA was extracted from hippocampal samples using the TRIzol method, and RNA quality was assessed using an Agilent 2100 bioanalyzer (Agilent, USA). mRNA was enriched using Oligo(dT) magnetic beads, fragmented, and reverse transcribed into double-stranded cDNA. Adapters were ligated to construct sequencing libraries, and PE150 sequencing was performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. on the NovaSeq X Plus platform. Raw data were filtered using FastP software for quality control, aligned to the rat reference genome using HISAT2, and gene expression levels were quantified using RSEM. Differentially expressed genes (DEGs) were screened using DESeq2 (screening threshold: |log2(FC)|>1, corrected P<0.05). Enrichment analysis using Goatools and Python scipy was performed on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG). WGCNA analysis was used to identify gene modules significantly associated with cognitive behavioral indicators.

[0024] This invention uses pseudo-time series analysis to explore key target genes in the development of POCD. The results show that, except for Cluster 4, the gene expression trajectories of the other four clusters (Cluster 1, Cluster 2, Cluster 3, and Cluster 5) highly coincide with the time window of POCD development. Figure 2(A) KEGG pathway enrichment analysis was performed on genes from the four key clusters mentioned above, and the results showed significant enrichment in the calcium signaling pathway, cAMP signaling pathway, and GnRH signaling pathway. Among these, the enrichment effect on the calcium signaling pathway was the most significant. Figure 2 (B in the middle).

[0025] Subsequently, the present invention extracted and plotted expression heatmaps of eight key genes involved in the calcium signaling pathway. Figure 2 (C in the text). The heatmap results showed that the expression levels of CaMK2δ, Tnnc1, and Adra1b were significantly upregulated on postoperative day 3 (POD 3). Quantitative fluorescence results (…) Figure 2 As confirmed in D), the expression changes of CaMK2δ are particularly dramatic, and this invention speculates that CaMK2δ is a key candidate pathogenic gene driving the occurrence and development of POCD.

[0026] Inhibition of Camk2δ can significantly reverse postoperative cognitive impairment in aged rats. To evaluate the protective effect of targeted inhibition of Camk2δ on postoperative cognitive function, this invention performed Morris water maze (MWM) and Y maze tests on rats in the Ctrl, POCD, and POCD+KN-93 groups after KN-93 intervention. Figure 3 Image A in the image represents the escape latency during the training period; the results of the MWM training period showed that there were no significant differences in the decreasing trend of latency and swimming speed among the groups of rats, thus excluding the interference of basic learning and motor ability. Figure 3 B and Figure 3 (D in the text). Behavioral test results showed that the POCD model group rats exhibited severe cognitive impairment, with a significantly prolonged MWM escape latency (approximately 2.3 times that of the control group), and a substantial reduction in target quadrant dwell time and platform crossing frequency. KN-93 intervention significantly reversed these deficiencies, resulting in a significantly shortened latency and increases in target quadrant dwell time and platform crossing frequency to 1.9 times and 3.6 times, respectively, that of the model group. Figure 3 E in Figure 3 F and Figure 3 (G in the text). Furthermore, the Y-maze experiment confirmed that KN-93 intervention significantly increased the spontaneous alternation rate, which was significantly reduced in the model group. Figure 3 H and Figure 3 (I in the text). In summary, inhibiting Camk2δ activity can effectively alleviate the impairment of spatial learning, reference memory, and working memory in POCD rats.

[0027] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a CaMK2δ-targeting agent in the preparation of a drug for improving or treating postoperative cognitive impairment; wherein the CaMK2δ-targeting agent is a CaMK2δ-inhibiting agent.

2. The application according to claim 1, characterized in that, The reagents for inhibiting CaMK2δ include reagents that inhibit CaMK2δ activity, reagents that inhibit CaMK2δ gene transcription, or reagents that inhibit CaMK2δ protein expression.

3. The application of CaMK2-targeting reagents in the preparation of drugs for improving or treating postoperative cognitive impairment; wherein the CaMK2-targeting reagent is a reagent that inhibits CaMK2.

4. The application according to claim 3, characterized in that, The reagents that inhibit CaMK2 include reagents that inhibit CaMK2 activity, reagents that inhibit CaMK2 gene transcription, or reagents that inhibit CaMK2 protein expression.

5. The application according to claim 1, characterized in that, The CaMK2 includes CaMK2α, CaMK2β, CaMK2γ, or CaMK2δ.

6. Application of KN-93 in the preparation of drugs to improve or treat postoperative cognitive impairment.

7. Application of reagents for detecting CaMK2 expression levels in the preparation of kits for diagnosing postoperative cognitive impairment.

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