Method, system and equipment for evaluating treatment effect of CGRP on epilepsy or brain injury caused by epilepsy

Abstract

The invention discloses a method, a system and equipment for evaluating the treatment effect of CGRP on epilepsy or brain injury caused by epilepsy. It is found for the first time that CGRP inhibits expression of P2RX7 by inhibiting a JAK-STAT1 pathway so as to play a role in treating epilepsy or brain injury caused by epilepsy. On the basis, the invention provides a method, a system and equipment for evaluating the treatment effect of the CGRP on the epilepsy or the brain injury caused by the epilepsy for the field, so that a doctor is assisted in evaluating the treatment effect of a patient suffering from the epilepsy or the brain injury caused by the epilepsy, and a clinician is guided to formulate an individualized treatment scheme for the patient.

Description

Technical Field

[0001] The present invention belongs to the field of bioinformatics. Specifically, the present invention relates to a method, system and device for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy. Background Art

[0002] Epilepsy is a chronic brain disease characterized by recurrent seizures. Triggered by abnormal discharges of brain neurons, seizures are recurrent and transient. Causes of epilepsy include muscle contractions, cerebral cortical developmental disorders, brain tumors, head trauma, central nervous system infections, and may also be genetic. Epilepsy is not limited to any age group, but is more common in children and the elderly.

[0003] Calcitonin gene-related peptide (CGRP) is a biologically active polypeptide with a molecular structure composed of 37 amino acids, a relative molecular mass of approximately 3800, and a biological half-life of approximately 18 minutes. It is homologous to the calcitonin gene and belongs to a family of neuropeptides. Current studies have found that CGRP has multiple biological activities: participating in the functional protection and repair of the cardiovascular system, the protection of the nervous system, and lung function, regulating bone damage and repair, and mediating various immune regulatory mechanisms in the body. It is of great significance in the diagnosis and treatment of diseases, but its role in epilepsy is unclear. Because the metabolism of the same drug varies greatly between patients, exploring the intrinsic mechanisms of drug treatment can provide a reliable method for evaluating the therapeutic effect of drugs against epilepsy, thereby helping clinicians adjust dosing regimens. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the present invention aims to provide a method, system and device for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy.

[0007] Furthermore, the method is performed by a computer and comprises the following steps:

[0008] Acquiring data: Obtaining expression data of p-JAK1, p-STAT1, and / or P2RX7 in samples from patients with epilepsy or brain damage caused by epilepsy after CGRP treatment;

[0009] Processing data: inputting the p-JAK1, p-STAT1, and / or P2RX7 expression data into a constructed evaluation model, wherein the evaluation model predicts the therapeutic effect of CGRP on patients with epilepsy or patients with brain damage caused by epilepsy based on the p-JAK1, p-STAT1, and / or P2RX7 expression data;

[0010] Output the results.

[0011] Furthermore, the p-JAK1 and p-STAT1 refer to phosphorylated JAK1 and phosphorylated STAT1, respectively.

[0012] Furthermore, the expression data of p-JAK1, p-STAT1 and / or P2RX7 include mRNA expression level data and protein expression level data.

[0013] Furthermore, the mRNA expression level data include, but are not limited to, data obtained by RT-PCR, qRT-PCR, in situ hybridization, and RNA sequencing.

[0014] Furthermore, the protein expression level data include but are not limited to data obtained by immunoblotting, immunohistochemistry, enzyme-linked immunosorbent assay, and mass spectrometry.

[0015] Furthermore, the steps for constructing the evaluation model are as follows:

[0016] Obtaining expression data of p-JAK1, p-STAT1, and / or P2RX7 from untreated epilepsy patients or patients with brain damage caused by epilepsy and patients with epilepsy or patients with brain damage caused by epilepsy after CGRP treatment; inputting the expression data of p-JAK1, p-STAT1, and / or P2RX7 into a machine learning algorithm to construct an evaluation model.

[0017] Furthermore, the step of constructing the evaluation model also includes verifying the model effectiveness, and the method of verifying the model effectiveness includes ROC curve.

[0018] Furthermore, the machine learning algorithm includes algorithm models developed using various development tools.

[0019] Furthermore, the development tools include but are not limited to TensorFlow, Scikit Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, and Spell.

[0020] Furthermore, the algorithm model includes but is not limited to linear regression model, logistic regression model, Lasso regression model, Ridge regression model, linear discriminant analysis model, nearest neighbor model, decision tree model, perceptron model, neural network model, support vector machine model, naive Bayes model, AdaBoost model, GBDT model, XGBoost model, LightGBM model, CatBoost model, and random forest model.

[0021] Furthermore, the evaluation model obtains results through the following criteria:

[0022] When the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are lower than a threshold value, a classification result is obtained that CGRP is effective in treating epilepsy patients or patients with brain damage caused by epilepsy; when the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are higher than a threshold value, a classification result is obtained that CGRP is ineffective in treating epilepsy patients or patients with brain damage caused by epilepsy.

[0023] In some embodiments of the present invention, the preset threshold is a representative value of normal samples from patients with epilepsy or brain damage caused by epilepsy, including but not limited to a maximum value, a third quartile, and a mean value. In some preferred embodiments of the present invention, the population sample includes more than 20 samples, for example, 30, 50, 80, 100, 150, 200, 300, 500, or more.

[0024] Furthermore, the patient includes humans and / or mammals.

[0025] Furthermore, the sample includes blood and / or tissue.

[0026] A second aspect of the present invention provides a system for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy.

[0027] Furthermore, the system comprises:

[0028] Data acquisition unit: used to obtain the expression data of p-JAK1, p-STAT1 and / or P2RX7 in samples from epilepsy patients or patients with brain damage caused by epilepsy after CGRP treatment;

[0029] A data classification unit is configured to classify and predict the data obtained by the data acquisition unit using the evaluation model obtained by the construction method described in the first aspect of the present invention, and obtain a classification result on whether CGRP is effective in treating epilepsy patients or patients with brain damage caused by epilepsy;

[0030] Result output unit: used to output classification results.

[0031] A third aspect of the present invention provides a computer device.

[0032] Furthermore, the device comprises:

[0033] A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy as described in the first aspect of the present invention is implemented.

[0034] A fourth aspect of the present invention provides a computer-readable storage medium.

[0035] Furthermore, the computer-readable storage medium stores a computer program, which, when executed by a processor, implements the method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy as described in the first aspect of the present invention.

[0036] A fifth aspect of the present invention provides the use of CGRP in the preparation of a drug for treating epilepsy or brain damage caused by epilepsy.

[0037] Furthermore, the CGRP plays a role in treating epilepsy or brain damage caused by epilepsy by inhibiting the JAK-STAT1 pathway and thereby inhibiting the expression of P2RX7.

[0038] Advantages and beneficial effects of the present invention:

[0039] This invention discovers for the first time that CGRP plays a role in treating epilepsy or epilepsy-induced brain damage by inhibiting the JAK-STAT1 pathway and, in turn, P2RX7 expression. Based on this, the invention provides a method, system, and device for evaluating the therapeutic efficacy of CGRP on epilepsy or epilepsy-induced brain damage, thereby assisting physicians in evaluating the therapeutic efficacy of epilepsy or epilepsy-induced brain damage patients and guiding clinicians in developing individualized treatment plans for their patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1A schematic flow chart of the method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy provided by the present invention;

[0041] Figure 2 Schematic diagram of the system structure for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy provided by the present invention;

[0042] Figure 3 A schematic diagram of the structure of the computer device provided by the present invention;

[0043] Figure 4 The figure shows that CGRP can reduce the frequency, grade and pathological damage of epileptic seizures induced by KA in mice; Figure 4 A is the result graph of epileptic seizure frequency in mice; Figure 4 B is the result of evaluating epileptic seizures in mice using the Racine scale; Figure 4 C is the expression result of inflammatory factors (IL-6, IL-1β, TNF-α) detected by qPCR; Figure 4 D. Figure 4 E are the results of HE and Nissl staining of pathological damage in the hippocampal CA1 and CA3 regions;

[0044] Figure 5 The figure shows the effect of CGRP treatment on the JAK-STAT1 pathway and the expression of P2RX7; Figure 5 A is the result of the effect of CGRP treatment on the expression of JAK-STAT1 pathway and P2RX7 in the hippocampus of KA-induced epileptic mice; Figure 5 B. Figure 5 C shows the effects of CGRP treatment on the expression of JAK-STAT1 pathway and P2RX7 in glutamate-induced HT22 neuronal cell lines and PC12 neuronal cell lines;

[0045] Figure 6 To enhance STAT1 transcriptional activity, CGRP can be blocked to inhibit the expression of P2RX7 protein; Figure 6 A is the result of enhancing STAT1 transcriptional activity on the expression of P2RX7 protein in the hippocampus of KA-induced epileptic mice; Figure 6 B. Figure 6 C is a graph showing the effect of enhancing STAT1 transcriptional activity on P2RX7 protein expression in glutamate-induced HT22 neuronal cell lines and PC12 neuronal cell lines. DETAILED DESCRIPTION

[0046] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0047] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or may be executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish between different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., and do not represent the order of precedence, nor do they limit "first" and "second" to be different types.

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making creative efforts shall fall within the scope of protection of the present invention.

[0049] Figure 1 This is a flow chart of a method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy provided by the present invention. Specifically, the method includes:

[0050] 101: Obtain data on the expression of p-JAK1, p-STAT1, and / or P2RX7 in samples from patients with epilepsy or brain damage caused by epilepsy after CGRP treatment;

[0051] In some embodiments of the present invention, the applicant, through extensive and in-depth research, found that the administration of CGRP to mice with kainate (KA)-induced epilepsy reduced the frequency, grade, and pathological damage of epileptic seizures, indicating that CGRP has the potential to treat epilepsy and brain damage caused by epilepsy. Further studies have found that CGRP exerts its effects by inhibiting the JAK-STAT1 pathway and thereby inhibiting the expression of P2RX7. Specifically, after CGRP treatment, the expression levels of p-JAK1, p-STAT1, and / or P2RX7 in epilepsy mouse models and glutamate-induced neuronal cell death models were significantly reduced. This suggests that p-JAK1, p-STAT1, and / or P2RX7 can be used as good markers to evaluate the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy.

[0052] In some embodiments, the patient can be human or non-human, and can include, for example, animal strains or species used as a "model system" for research purposes. Likewise, patients can include adults or adolescents (e.g., children). In addition, a patient can refer to any living organism that benefits from the Meconopsis described herein, preferably a mammal (e.g., human or non-human). Examples of mammals include, but are not limited to, any member of the class of mammals: humans, non-human primates (e.g., chimpanzees), and other apes and monkeys; livestock, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; experimental animals include rodents, such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, etc.

[0053] As used in the context of the present invention, the term "sample" refers to a composition obtained from or derived from a patient / subject that contains cells and / or other molecular entities to be characterized and / or identified based on, for example, physical, biochemical, chemical and / or physiological characteristics. For example, a sample refers to any sample derived from a patient / subject that is expected or known to contain cells and / or molecular entities to be characterized. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell cultures, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, pancreatic juice, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tissue culture fluid, tissue extracts, homogenized tissue, cell extracts, and combinations thereof. In specific embodiments of the present invention, the sample is a tissue or a cell.

[0054] In one embodiment of the present invention, the applicant found that CGRP can reduce the frequency, grade, and pathological damage of KA-induced epileptic seizures in mice. The experimental mice were divided into three groups. Sham group: an equal amount of normal saline was injected into the hippocampus of the mice; KA group: 1uL (0.3ug) KA was injected into the hippocampus of the mice; KA+CGRP group: 1uL KA was injected into the hippocampus of the mice, and CGRP was injected intraperitoneally at the same time. The epileptic seizure status of the mice was continuously recorded for 24 hours using a video monitoring and acquisition system. The results are as follows: Figure 4 As shown in the figure, compared with the KA+Saline (normal saline) group, the frequency of epileptic seizures in mice in the KA+CGRP group was significantly reduced and there was a significant difference, which indicates that CGRP significantly reduced the frequency of epileptic seizures in mice induced by kainate (KA) ( Figure 4 A). The Racine scale was used to evaluate the epileptic seizures in mice. Figure 4As shown in Figure B, compared with the KA+Saline group, the epilepsy level in the KA+CGRP group was reduced by about one-half, indicating that CGRP can reduce the grade of epileptic seizures induced by kainate (KA) in mice. After anesthetizing the mice and performing cardiac perfusion, the hippocampal tissue of the mice was carefully dissected out, TRIzol was added, and the tissue was homogenized using a tissue grinder. Tissue RNA was extracted and the expression of inflammatory factors was examined by qPCR. The degree of inflammation in the mouse hippocampus was evaluated using inflammatory factors (IL-1β, IL-6, TNF-α) in the hippocampus. The results are shown in Figure 2. Figure 4 As shown in C, CGRP can reduce the degree of inflammation in the hippocampus of mice with epilepsy induced by kainate (KA). HE and Nissl staining results also showed that CGRP can reduce the pathological damage of CA1 and CA3 regions of the hippocampus in mice with epilepsy induced by kainate (KA). Figure 4 D. Figure 4 E).

[0055] In one embodiment of the present invention, the applicant discovered that CGRP can inhibit the JAK-STAT1 pathway and thereby inhibit the expression of P2RX7 in a KA-induced epilepsy mouse model and a glutamate-induced neuronal cell injury model. Western blot experiments were performed to detect the expression levels of p-JAK1, p-STAT1, and P2RX7 in mice in the sham, KA+saline, and KA+CGRP groups. The results were as follows: Figure 5 As shown in A, compared with the sham operation (Sham) group, the expression levels of p-JAK1, p-STAT1 and P2RX7 in the hippocampal tissue of mice in the KA+Saline (normal saline) group were significantly increased, while the expression levels of p-JAK1, p-STAT1 and P2RX7 in the hippocampal tissue of mice were significantly decreased after the administration of CGRP, indicating that CGRP can inhibit the JAK-STAT1 pathway and thus inhibit the expression of P2RX7 to exert its effect. Further, the applicant verified this in an in vitro cell experiment. The neuronal cell lines HT22 cells and PC12 cells were plated and divided into 3 groups: Control group: no treatment; Glutamate group: Glutamate was added to the culture medium; Glutamate+CGRP group: Glutamate and CGRP were added to the culture medium. Afterwards, the expression of p-JAK1, p-STAT1 and P2RX7 in different groups was detected by Western blot experiment. The results are shown in Figure 2. Figure 5As shown in Figure B, compared with the normal group cells, the expression levels of p-JAK1, p-STAT1 and P2RX7 in the Glutamate model injury group cells were significantly increased, while the expression levels of p-JAK1, p-STAT1 and P2RX7 in the mouse hippocampus tissue were significantly decreased after the administration of CGRP, further indicating that CGRP exerts its effect by inhibiting the JAK-STAT1 pathway and thereby inhibiting the expression of P2RX7.

[0056] In one embodiment of the present invention, the applicant further demonstrated the relationship between STAT1 transcriptional activity and P2RX7 protein expression levels. 2-NP is a selective STAT1 transcription enhancer that can enhance STAT1 transcriptional activity by activating the JAK-STAT signaling pathway. Therefore, to demonstrate the relationship between STAT1 transcriptional activity and P2RX7 protein expression levels, we added 2-NP to epilepsy mouse models and cell models. The results are shown in Figure 2. Figure 6 As shown in the results, the addition of STAT1 transcription enhancer 2-NP alleviated the inhibition of P2RX7 protein expression by CGRP in both epilepsy mouse model and cell model, indicating that enhancing STAT1 transcription activity can block the inhibition of P2RX7 protein expression by CGRP, further indicating that CGRP inhibits P2RX7 expression by inhibiting the JAK-STAT1 pathway.

[0057] 102: Processing data, inputting the expression data of p-JAK1, p-STAT1 and / or P2RX7 into a constructed evaluation model, wherein the evaluation model predicts the therapeutic effect of CGRP on epilepsy patients or patients with brain damage caused by epilepsy based on the expression data of p-JAK1, p-STAT1 and / or P2RX7;

[0058] In some embodiments of the present invention, the method of constructing the evaluation model is known to those skilled in the art, and the step of associating the expression level of p-JAK1, p-STAT1 and / or P2RX7 with a certain possibility or risk can be implemented and realized in different ways.

[0059] In the context of the present invention, the term "machine learning" refers to the use of computers to simulate or implement human learning activities. Technicians typically use different development tools to build machine learning algorithm models. The development tools include but are not limited to TensorFlow, Scikit Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, Spell. The algorithm models include but are not limited to linear regression models, logistic regression models, Lasso regression models, Ridge regression models, linear discriminant analysis models, nearest neighbor models, decision tree models, perceptron models, neural network models, support vector machine models, naive Bayes models, AdaBoost models, GBDT models, XGBoost models, LightGBM models, CatBoost models, or random forest models.

[0060] In one embodiment, after the evaluation model is constructed, the ROC curve analysis can be used to evaluate the effectiveness of the model.

[0061] An ROC curve is a plot of the true positive rate (sensitivity) of a test against the false positive rate (100% minus specificity) of the test. It is useful for depicting the performance of a particular feature when distinguishing between two populations. Typically, feature data is selected in ascending order across the entire population based on the value of a single feature. Then, for each value of that feature, the true positive and false positive rates of the data are calculated. The true positive rate is determined by counting the number of cases with a value above that feature and dividing it by the total number of cases. The false positive rate is determined by counting the number of controls with a value above that feature and dividing it by the total number of controls. While this definition refers to situations where a feature is elevated in cases compared to controls, it also applies to situations where a feature is low in cases compared to controls (in which case, samples with a value below that feature would be counted). ROC curves can be generated for individual features, as well as for other individual outputs. For example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single summed value, which can then be plotted in an ROC curve. Additionally, any combination of multiple features where the combination is derived from a single output value can be plotted in an ROC curve.

[0062] 103: Output the result.

[0063] In some embodiments of the present invention, when the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are lower than a threshold value, a classification result is obtained that CGRP is effective in treating patients with epilepsy or patients with brain damage caused by epilepsy; when the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are higher than a threshold value, a classification result is obtained that CGRP is ineffective in treating patients with epilepsy or patients with brain damage caused by epilepsy.

[0064] In some embodiments of the present invention, the preset threshold is a representative value of normal samples from patients with epilepsy or brain damage caused by epilepsy, including but not limited to a maximum value, a third quartile, and a mean value. In some preferred embodiments of the present invention, the population sample includes more than 20 samples, for example, 30, 50, 80, 100, 150, 200, 300, 500, or more.

[0065] Figure 2 This is a schematic diagram of the system structure for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy provided by the present invention.

[0066] The system is programmed or otherwise configured to include a data acquisition unit 201, a data classification unit 202, and a result output unit 203:

[0067] Data acquisition unit: used to obtain the expression data of p-JAK1, p-STAT1 and / or P2RX7 in samples from epilepsy patients or patients with brain damage caused by epilepsy after CGRP treatment;

[0068] A data classification unit is configured to classify and predict the data obtained by the data acquisition unit using the evaluation model obtained by the construction method described in the first aspect of the present invention, and obtain a classification result on whether CGRP is effective in treating epilepsy patients or patients with brain damage caused by epilepsy;

[0069] Result output unit: used to output classification results.

[0070] The system may be the user's electronic device or a computer system located remotely from the electronic device.

[0071] Figure 3 This is a schematic structural diagram of the computer device provided by the present invention.

[0072] The computer device 300 includes a processor 301 and a memory 302 coupled to the processor 301. The memory 302 stores program instructions. When the program instructions are executed by the processor 301, the processor 301 executes the above-mentioned method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy.

[0073] The processor 301 may also be referred to as a CPU (Central Processing Unit). The processor 301 may be an integrated circuit chip having signal processing capabilities. The processor 301 may also be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic device, a discrete gate or transistor logic device, or a discrete hardware component. The general-purpose processor may be a microprocessor or any conventional processor.

[0074] The computer device 300 may be a mobile electronic device.

[0075] It should be understood that the systems, devices, and methods described herein can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the module division is merely a logical functional division, and actual implementations may employ other division methods, such as combining or integrating multiple modules or components into another system, or omitting or disabling certain features. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be an indirect coupling or communication connection between devices or modules via some interface, which may be electrical, mechanical, or other forms.

[0076] The modules described as separate components may or may not be physically separate, and the components shown as modules may or may not be physical units, that is, they may be located in one place or distributed across multiple network elements. Some or all of these units may be selected to achieve the purpose of this embodiment according to actual needs.

[0077] In addition, the functional modules in various embodiments of the present invention may be integrated into a single processing module, or each module may exist physically separately, or two or more modules may be integrated into a single module. The aforementioned integrated modules may be implemented in the form of hardware or software functional modules.

[0078] Example CGRP treats epileptic brain damage by inhibiting the JAK-STAT1-P2RX7 pathway

[0079] 1. Experimental instruments, reagents and materials

[0080] 1. Instruments

[0081] A high-speed cryogenic centrifuge and a high-speed cryogenic tissue grinder were purchased from Wuhan Seville Biotechnology Co., Ltd.; a microinjector and a brain stereotaxic apparatus were purchased from Shenzhen Reward Life Science Co., Ltd.; and a chemiluminescence imaging system was purchased from Shanghai Qinxiang Scientific Instrument Co., Ltd.

[0082] 2. Reagents

[0083] Kainate (KA) and CGRP were purchased from MCE, USA; β-actin and P2RX7 antibodies were purchased from Wuhan Abotek Biotechnology Co., Ltd.; JAK1, p-JAK1, STAT1, and p-STAT1 antibodies were purchased from Cell Signaling Technology, USA.

[0084] 3. Experimental mice

[0085] 6- to 8-week-old male C57BL / 6J mice were purchased from Beijing Spefoc Biotechnology Co., Ltd.

[0086] 2. Experimental Methods

[0087] 1. Epilepsy mouse model and construction

[0088] 1) Anesthesia: Inject 1% sodium pentobarbital (10 μL / g) intraperitoneally into C57BL / 6J mice. Pinch the mouse's tail with your finger. If there is no response, anesthesia is successful. This should last approximately 10 minutes.

[0089] 2) Fixation: After successful anesthesia, place the mouse on a digital stereotaxic apparatus. Hang the mouse's teeth on the front incisor clamp of the apparatus. Keep the mouse's head and body in a straight line and on a horizontal plane. Insert the two fixing rods into the mouse's ears to fix the head so that it does not shake. Adjust the mouse's head to the center position. Pull the mouse's tongue outward to avoid airway obstruction. Always monitor the mouse's breathing and heart rate.

[0090] 3) Skin preparation: Use curved scissors to trim the mouse hair 1 cm on each side of the sagittal line and disinfect the exposed scalp with iodine.

[0091] 4) Expose the bregma: Cut the skin along the sagittal line to a size of about 2 cm. The bregma will be visible. Wipe the bregma with 1% hydrogen peroxide and remove the periosteum.

[0092] 5) Positioning: Place a microsyringe containing 1 μL of KA on a digital stereotaxic instrument. Move the syringe needle to the bregma point. Using the bregma point as the origin, move the needle tip so that it is 1.8 mm behind the bregma point and 2 mm to the right of the sagittal line. Mark this point with a marker.

[0093] 6) Drilling: Use an electric miniature handheld cranial drill to drill the skull at the marked point and stop after the dura mater is exposed.

[0094] 7) Brain microinjection: Move the microinjector needle back to the marked point and advance the needle at a rate of 1 mm / min. Stop advancing after approximately 2.3 mm and inject 1 μL of KA or saline into the hippocampus at a rate of 0.02 μL / min. Keep the mouse scalp moist with a cotton ball dipped in saline. After the injection, leave the needle in place for 5 minutes and slowly remove the syringe needle to prevent backflow of the injection solution.

[0095] 8) Skin suturing: After observing that there is no active bleeding, suture the mouse scalp and disinfect the scalp with iodine.

[0096] 2. Experimental Design

[0097] The experimental mice were divided into three groups: sham group: mice were injected with an equal volume of normal saline into the hippocampus; KA group: mice were injected with 1uL (0.3ug) KA into the hippocampus; KA+CGRP group: mice were injected with 1uL KA into the hippocampus and CGRP was injected intraperitoneally.

[0098] HT22 cells and PC12 cells were plated and divided into three groups: Control group: no treatment; Glutamate group: Glutamate was added to the culture medium; Glutamate+CGRP group: Glutamate and CGRP were added to the culture medium.

[0099] 3. Evaluation of epileptic seizure grading in mice

[0100] One hour after intracerebroventricular injection of KA, mice were assessed for seizure activity using the Racine scale. The Racine scale includes the following criteria: Grade 0: no seizures; Grade 1: oral and facial twitching, including jaw clonus; Grade 2: Grade 1 plus rhythmic nodding; Grade 3: Grade 2 plus unilateral forelimb twitching; Grade 4: bilateral forelimb twitching with standing; and Grade 5: generalized tonic-clonic convulsions with falling while standing. Sustained occurrence of Grade 4 or higher (≥2 times within 30 minutes) was considered status epilepticus.

[0101] 4. Detection of epileptic seizure frequency in mice

[0102] After KA was injected into the lateral ventricle of mice, the epileptic seizure status of the mice was continuously recorded within 24 hours using a video monitoring and acquisition system, with the presence of a seizure being recorded as 1 and the absence of a seizure being recorded as 0.

[0103] 5. Detection of inflammatory factor expression in mouse hippocampus tissue

[0104] After anesthetizing mice and performing cardiac perfusion, the hippocampus was carefully dissected out. TRIzol was added and homogenized using a tissue grinder. Tissue RNA was extracted and analyzed for the expression of inflammatory factors using qPCR.

[0105] 6. HE staining of mouse hippocampus tissue

[0106] After anesthetizing mice and performing cardiac perfusion, the brain tissue was carefully excised and fixed in 4% paraformaldehyde for 48 hours. The sections were dehydrated, embedded, frozen, sectioned, stained with hematoxylin, differentiated, bluing, and eosin stained, dehydrated, and mounted. The sections were then observed under a light microscope and photographed for preservation.

[0107] 7. Nissl staining of mouse hippocampal tissue

[0108] After anesthetizing mice and performing cardiac perfusion, the brain tissue was carefully excised and fixed in 4% paraformaldehyde for 48 hours. The brain tissue was then dehydrated, embedded, frozen sectioned, stained with toluidine blue, differentiated, dehydrated, and mounted. The tissue was then observed under a light microscope and photographed for preservation.

[0109] 8. Western blot experiment

[0110] (1) Protein sample preparation: Add RIPA lysis buffer (with 1% protease inhibitor) to the tissue or cell sample and lyse on ice for 20 min, using a shaker to increase lysis efficiency. Centrifuge at 12,000 rpm at 4°C for 15 min, and collect the supernatant. Then, add an appropriate volume of 5× loading buffer and boil for 10 min to obtain the WB protein sample.

[0111] (2) Immunoblotting: Separate protein samples using SDS-PAGE gel. After electrophoresis, transfer to a PVDF membrane. Incubate with 2.5% blocking buffer for 2 hours, then incubate with the primary antibody at 4°C overnight. The next day, wash three times with TBST, incubate with the corresponding secondary antibody at room temperature for 2 hours, and then wash the membrane three times. Then, add ECL luminescent solution in a developer to develop the image.

[0112] 3. Experimental Results

[0113] The epileptic seizure status of mice was continuously recorded within 24 hours by a video monitoring and acquisition system. Figure 4 As shown in the figure, compared with the KA+Saline (normal saline) group, the frequency of epileptic seizures in mice in the KA+CGRP group was significantly reduced and there was a significant difference, which indicates that CGRP significantly reduced the frequency of epileptic seizures in mice induced by kainate (KA) ( Figure 4 A). The Racine scale was used to evaluate the epileptic seizures in mice. Figure 4As shown in Figure B, compared with the KA+Saline group, the epilepsy level in the KA+CGRP group was reduced by about one-half, indicating that CGRP can reduce the grade of epileptic seizures induced by kainate (KA) in mice. After anesthetizing the mice and performing cardiac perfusion, the hippocampal tissue of the mice was carefully dissected out, TRIzol was added, and the tissue was homogenized using a tissue grinder. Tissue RNA was extracted and the expression of inflammatory factors was examined by qPCR. The degree of inflammation in the mouse hippocampus was evaluated using inflammatory factors (IL-1β, IL-6, TNF-α) in the hippocampus. The results are shown in Figure 2. Figure 4 As shown in C, CGRP can reduce the degree of inflammation in the hippocampus of mice with epilepsy induced by kainate (KA). HE and Nissl staining results also showed that CGRP can reduce the pathological damage of CA1 and CA3 regions of the hippocampus in mice with epilepsy induced by kainate (KA). Figure 4 D. Figure 4 E).

[0114] Western blot experiments were performed to detect the expression levels of p-JAK1, p-STAT1, and P2RX7 in mice in the sham, KA+Saline, and KA+CGRP groups. Figure 5 As shown in A, compared with the sham operation (Sham) group, the expression levels of p-JAK1, p-STAT1 and P2RX7 in the hippocampus of mice in the KA+Saline (normal saline) group were significantly increased, while the expression levels of p-JAK1, p-STAT1 and P2RX7 in the hippocampus of mice after CGRP administration were significantly decreased, indicating that CGRP can inhibit the JAK-STAT1 pathway and thus inhibit the expression of P2RX7. At the same time, Western blot experiments were used to detect the expression of p-JAK1, p-STAT1 and P2RX7 in different cell groups. The results are shown in Figure 5 As shown in Figure B, compared with the normal group cells, the expression levels of p-JAK1, p-STAT1 and P2RX7 in the Glutamate model injury group cells were significantly increased, while the expression levels of p-JAK1, p-STAT1 and P2RX7 in the mouse hippocampus tissue were significantly decreased after the administration of CGRP, further indicating that CGRP exerts its effect by inhibiting the JAK-STAT1 pathway and thereby inhibiting the expression of P2RX7.

[0115] To further demonstrate the relationship between STAT1 transcriptional activity and P2RX7 protein expression levels, we added 2-NP (STAT1 transcription enhancer) to epilepsy mouse models and cell models. Figure 6As shown in the results, the addition of STAT1 transcription enhancer 2-NP alleviated the inhibition of P2RX7 protein expression by CGRP in both epilepsy mouse model and cell model, indicating that enhancing STAT1 transcription activity can block the inhibition of P2RX7 protein expression by CGRP, further indicating that CGRP inhibits P2RX7 expression by inhibiting the JAK-STAT1 pathway.

[0116] The above is only an implementation method of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the description and drawings of this application, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy, characterized in that: The method is performed by a computer and comprises the following steps: Acquiring data: Obtaining expression data of p-JAK1, p-STAT1, and / or P2RX7 in samples from patients with epilepsy or brain damage caused by epilepsy after CGRP treatment; Processing data: inputting the p-JAK1, p-STAT1, and / or P2RX7 expression data into a constructed evaluation model, wherein the evaluation model predicts the therapeutic effect of CGRP on patients with epilepsy or patients with brain damage caused by epilepsy based on the p-JAK1, p-STAT1, and / or P2RX7 expression data; Output the results.

2. The method according to claim 1, characterized in that The steps for constructing the evaluation model are as follows: Obtaining expression data of p-JAK1, p-STAT1, and / or P2RX7 from untreated epilepsy patients or patients with brain damage caused by epilepsy and CGRP-treated epilepsy patients or patients with brain damage caused by epilepsy; The expression data of p-JAK1, p-STAT1 and / or P2RX7 were input into the machine learning algorithm to construct the evaluation model.

3. The method according to claim 2, characterized in that The step of constructing the evaluation model also includes verifying the model effectiveness, and the method of verifying the model effectiveness includes ROC curve.

4. The method according to claim 2, characterized in that The machine learning algorithm includes an algorithm model developed using various development tools; Preferably, the development tools include but are not limited to TensorFlow, Scikit Learn, PyTorch, OpenNN, RapidMiner, Azure Machine Learning, Apache Mahout, Shogun, KNIME, Vertex AI, H2Oai, Anaconda, Keras, Tableau, Fast.ai, Catalyst, Amazon ML, MLJAR, Spell; Preferably, the algorithm model includes but is not limited to a linear regression model, a logistic regression model, a Lasso regression model, a Ridge regression model, a linear discriminant analysis model, a nearest neighbor model, a decision tree model, a perceptron model, a neural network model, a support vector machine model, a naive Bayes model, an AdaBoost model, a GBDT model, an XGBoost model, a LightGBM model, a CatBoost model, and a random forest model.

5. The method according to claim 1, wherein The evaluation model obtains results through the following criteria: When the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are lower than a threshold value, a classification result is obtained that CGRP is effective in treating epilepsy patients or patients with brain damage caused by epilepsy; when the expression levels of p-JAK1, p-STAT1 and / or P2RX7 are higher than a threshold value, a classification result is obtained that CGRP is ineffective in treating epilepsy patients or patients with brain damage caused by epilepsy.

6. A system for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy, characterized in that: The system comprises: Data acquisition unit: used to obtain the expression data of p-JAK1, p-STAT1 and / or P2RX7 in samples from epilepsy patients or patients with brain damage caused by epilepsy after CGRP treatment; A data classification unit: configured to classify and predict the data obtained by the data acquisition unit using the evaluation model obtained by the construction method according to any one of claims 2 to 4, and obtain a classification result of whether CGRP is effective in treating epilepsy patients or patients with brain damage caused by epilepsy; Result output unit: used to output classification results.

7. A computer device, characterized in that: The device comprises: A memory and a processor, wherein the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, the method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy according to any one of claims 1 to 5 is implemented.

8. A computer-readable storage medium, characterized in that The computer-readable storage medium stores a computer program, which, when executed by a processor, implements the method for evaluating the therapeutic effect of CGRP on epilepsy or brain damage caused by epilepsy according to any one of claims 1 to 5.

9. Use of CGRP in the preparation of drugs for treating epilepsy or brain damage caused by epilepsy.

10. The use according to claim 9, characterized in that The CGRP plays a role in treating epilepsy or brain damage caused by epilepsy by inhibiting the JAK-STAT1 pathway and then inhibiting the expression of P2RX7.

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