Method for researching action mechanism of cornu cervi pantotrichum polypeptide on mild cognitive impairment based on Wnt / beta-catenin signal channel

Abstract

The invention discloses a method for researching a mild cognitive impairment action mechanism of a cornu cervi pantotrichum polypeptide based on a Wnt / beta-catenin signal channel, relates to the technical field of mechanism analysis, and solves the problem of difficulty in realizing spanning from basic research to clinical transformation in the prior art. Randomly dividing the rats into five groups of blank control, model, positive control and VAP high / low dose, continuously performing intragastric administration on each group for 18 days, and feeding distilled water into the blank and model groups; 2 mg / kg scopolamine is injected into abdominal cavities of the model group after 18 days and 2 hours, and normal saline is injected into the blank group; the scopolamine model is used for measuring the 2min static / motion time of the rat by using an open field experiment, and the unilateral common carotid artery ligation model is used for measuring the escape latency / platform penetration times by using a water maze; the method comprises the following steps: taking blood, separating a hippocampal region, and carrying out HE dyeing, ELISA and Western blot; sPSS 21.0 analysis and single factor variance analysis are used for evaluating the difference and Plt; 0.05 or Plt; 0.01 has statistical significance.

Description

[0001] A research method for studying the mechanism of action of deer antler peptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment. Technical Field

[0002] This invention relates to the field of mechanism analysis technology, specifically to a research method for studying the mechanism of action of deer antler peptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment. Background Technology

[0003] Mild cognitive impairment (MCI) is a transitional stage from normal brain aging to Alzheimer's disease (AD), and its core symptom is cognitive decline. Depending on the cause or the location of brain damage, it can affect one or more of the following: memory, executive function, language, use, visuospatial structural skills, leading to corresponding clinical symptoms. Since AD ​​is irreversible, early intervention in the development of MCI is of great significance in reducing the incidence of AD. Based on the clinical manifestations of MCI and other factors, the ideal characteristics of an animal model of MCI are old age, mild memory impairment, mild neuropathological changes, cholinergic system changes, and cerebrovascular changes [3]. The cholinergic system in the brain of MCI patients is disordered, and the ACh content is reduced, which is the main reason for memory decline. By destroying the cholinergic system in the brain of animals, the pathological characteristics of cholinergic decline in MCI can be reproduced to create an animal model of MCI. Vascular cognitive impairment (VCI) is an acquired intellectual disability syndrome caused by various cerebrovascular factors, particularly cumulative damage to brain tissues such as the hippocampus and cortex. Its pathological features include multiple cerebral infarctions and recurrent cerebral ischemia. Single common carotid artery ligation can replicate an early-stage animal model of VCI, namely a model of mild vascular cognitive impairment. Misfolding and pathological changes of Tau protein are fundamental to neurodegenerative diseases. Highly phosphorylated Tau protein, due to structural alterations, can form oligomers, which further develop into paired helical filaments (PHFs) with a double-helical ligation structure. PHFs further form neurofibrillary tangles, leading to synaptic annulment and cognitive decline. Okada acid (OA) is a phosphatase inhibitor that can be rapidly metabolized, thereby altering the phosphorylation-dephosphorylation rate in the body. Wistar male rats are typically used. OA is injected into the lateral ventricle via a micropump, causing phosphorylated Tau protein to exhibit a double-helix filamentous pathological change, leading to β-amyloid protein deposition and subsequent neuronal degeneration, synaptic loss, and memory impairment [6]. D-gal is a reducing sugar in the human body that can be metabolized into glucose at normal physiological concentrations. However, at high concentrations, D-gal is converted into galactitol by aldose reductase. Galactitol is then oxidized by activated galactose dehydrogenase, producing oxidation products hydrogen peroxide and aldehydes. This process generates three types of reactive oxygen species (ROS). Excessive ROS further attack cells, causing damage to neurons, resulting in learning and memory impairment and aging in animals.D-gal-induced aging or MCI animal models are widely used in pharmacology, pathology and other studies due to their simple operation, high reproducibility, minimal trauma and comprehensive mechanism. The main mechanisms of D-gal-induced MCI models also include: cholinergic system damage, apoptosis, inflammatory response, reduction of peripheral benzodiazepine receptors and immune-related mechanisms.

[0004] The PI3K / AKT signaling pathway is widely present in various cells and is one of the main pathways for neural survival signal transduction. It is a classic anti-apoptotic and pro-survival signal transduction pathway and a key signaling pathway for regulating brain cognitive function. The Wnt signaling pathway plays a role in maintaining the proliferation of tissue stem cells, and the expression of pathway-related proteins may affect the aging process. The Wnt2 signaling pathway is also involved in the development of the central nervous system and has the function of regulating synaptic plasticity and memory. Studies have shown that AD in animal models is related to Wnt pathway damage, and Wnt activation can repair learning and memory abilities and improve cognitive dysfunction. It is worth noting that GSK3β is a key regulatory hub for the Wnt / β-catenin and PI3K / AKT signaling pathways, and changes in its expression can prove the close regulatory connection between the two pathways. Deer antler is a traditional and precious Chinese medicinal material, which is the unossified, densely haired young antler of male sika deer (Cervus nippon Temminck) or red deer (Cervuse laphhus Linnaeus). Deer antler contains various active ingredients, including antler polypeptides, steroidal compounds, proteins, polysaccharides, and inorganic salts. Among these, antler polypeptide (VAP) is the main active ingredient, containing multiple bioactive factors that regulate the nervous, motor, immune, and circulatory systems. The increasing aging population has led to a year-on-year increase in the rate of cognitive impairment. Studies have shown that VAP can protect neurons and damaged nerve cells, reducing the incidence of neurodegenerative diseases such as Alzheimer's disease, and delaying or mitigating the development of these diseases by regulating related pathways and proteins in in vitro and in vivo models.

[0005] Currently, existing research has only confirmed that VAP can delay the progression of MCI, but it has not verified its mechanism of action based on clinically relevant composite pathological models, nor has it clarified the core role and weight of the Wnt / β-catenin pathway in VAP intervention. As a result, the clinical application of VAP lacks clear mechanistic support, making it difficult to achieve the leap from basic research to clinical translation. Summary of the Invention

[0006] To address the problem that existing technologies have not clearly defined the core role and weight of the Wnt / β-catenin pathway in VAP intervention, resulting in a lack of clear mechanistic support for the clinical application of VAP and making it difficult to achieve the leap from basic research to clinical translation, this invention proposes a research method for the mechanism of action of deer antler peptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment.

[0007] The specific technical solution of the present invention is as follows: This invention provides a method for studying the mechanism of action of deer antler polypeptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment, characterized by comprising the following steps: S1. Wistar rats were acclimatized for 7 days and then randomly divided into a blank control group, a model group, a positive control group, a high-dose VAP group, and a low-dose VAP group using a randomized parallel control method. Each group was administered the drug by gavage for 18 consecutive days. The blank control group and the model group were given an equal volume of distilled water. S2. Two hours after administration on day 18, the model group, positive control group and VAP group were injected intraperitoneally with scopolamine 2 mg / kg, while the blank control group was injected with an equal volume of normal saline. S3, Unilateral common carotid artery ligation: After adaptive feeding, except for the blank control group, the left common carotid artery of the remaining rats was ligated after anesthesia, and the drugs were administered 3 days after the operation. S4. The rats in the intraperitoneal injection scopolamine model were tested using the open field test to determine their resting time and movement time within 2 minutes; the rats in the unilateral common carotid artery ligation model were tested using the Morris water maze to determine their escape latency and number of times they crossed platforms within 3 minutes. S5. After the behavioral experiment, the rats were anesthetized and blood was collected. They were then sacrificed by cervical dislocation and the hippocampal tissue was separated. S6 and HE staining were used to observe pathological changes in the hippocampus; serum SOD, MDA, and BDNF levels were detected by ELISA; and the expression of PI3K, AKT, and Bad-related proteins in the hippocampus was detected by Western blot. S7. Using SPSS 21.0 software, express the data as mean ± standard deviation, and use one-way ANOVA to assess differences. P < 0.05 or P < 0.01 is considered statistically significant.

[0008] Compared with the prior art, the specific beneficial effects of the present invention are as follows: 1. This invention utilizes intraperitoneal injection of scopolamine and unilateral carotid artery ligation to replicate different MCI animal models, demonstrating that VAP may exert an antioxidant effect by increasing SOD content and inhibiting MDA production in model rats; it also produces a neuroprotective effect by increasing BDNF, NGF, and VEGF content in model rats, improving the learning and memory abilities of different MCI model rats. The mechanism of action may be related to the regulation of the expression of factors related to the PI3K / AKT signaling pathway.

[0009] 2. This invention utilizes an OA-induced HT22 cell damage model to demonstrate that VAP can enhance cell viability in the OA-induced HT22 cell damage model, increase SOD and GSH-PX activity in HT22 cells, inhibit MDA production, upregulate PI3K and AKT protein expression, and downregulate Bad, Bax, and Caspase-3 protein expression. This elucidates that VAP may antagonize OA-induced damaged HT22 cells through antioxidant and anti-apoptotic pathways, and its mechanism of action may be related to the regulation of the PI3K / AKT signaling pathway.

[0010] 3. This invention utilizes a rat model of brain aging established by D-gal and an HT22 cell injury model to demonstrate that the repair mechanism of damage caused by D-gal by VAP may involve affecting tissue oxidation and inflammatory factors, adjusting the expression of proteins such as BAX, Bcl-2, Caspase-3, and Caspase-9, and generating damage such as antioxidant, inflammatory, and apoptotic effects. The mechanism of action may be related to the activation of the Wnt / β-catenin signaling pathway.

[0011] 4. In the above-mentioned animal and cell models, the expression level of GSK-3β was upregulated and decreased after VAP treatment. This invention confirms that the PI3K / AKT and Wnt / β-catenin signaling pathways can achieve a unified mechanism through the shared GSK3β-β-catenin-Bcl2 regulatory axis, and proves that VAP exerts a neuroprotective effect by regulating the Wnt pathway in different MCI models.

[0012] In summary, this invention demonstrates that VAP can exert a therapeutic effect in different experimental models of MCI, and its mechanism of action involves regulating the PI3K / AKT and Wnt / β-catenin signaling pathways to alleviate neuroinflammation, oxidative stress, and apoptosis in MCI models. Detailed Implementation

[0013] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0014] Example 1. This embodiment provides a method for studying the mechanism of action of deer antler polypeptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment, characterized by the following steps: S1. Wistar rats were acclimatized for 7 days and then randomly divided into a blank control group, a model group, a positive control group, a high-dose VAP group, and a low-dose VAP group using a randomized parallel control method. Each group was administered the drug by gavage for 18 consecutive days. The blank control group and the model group were given an equal volume of distilled water. S2. Two hours after administration on day 18, the model group, positive control group and VAP group were injected intraperitoneally with scopolamine 2 mg / kg, while the blank control group was injected with an equal volume of normal saline. S3, Unilateral common carotid artery ligation: After adaptive feeding, except for the blank control group, the left common carotid artery of the remaining rats was ligated after anesthesia, and the drugs were administered 3 days after the operation. S4. The rats in the intraperitoneal injection scopolamine model were tested using the open field test to determine their resting time and movement time within 2 minutes; the rats in the unilateral common carotid artery ligation model were tested using the Morris water maze to determine their escape latency and number of times they crossed platforms within 3 minutes. S5. After the behavioral experiment, the rats were anesthetized and blood was collected. They were then sacrificed by cervical dislocation and the hippocampal tissue was separated. S6 and HE staining were used to observe pathological changes in the hippocampus; serum SOD, MDA, and BDNF levels were detected by ELISA; and the expression of PI3K, AKT, and Bad-related proteins in the hippocampus was detected by Western blot. S7. Using SPSS 21.0 software, express the data as mean ± standard deviation, and use one-way ANOVA to assess differences. P < 0.05 or P < 0.01 is considered statistically significant.

[0015] Experimental Results: Compared with the Control group, the Model group showed significantly increased stationary time and corner dwell time within 2 minutes (P < 0.01), and significantly decreased movement time, behavioral path, center dwell time, and edge dwell time (P < 0.01). Compared with the Model group, the PIR, VAPH, and VAPL groups showed significantly decreased stationary time (P < 0.01), and significantly increased movement time and behavioral path (P < 0.01); the PIR and VAPH groups showed significantly increased center dwell time (P < 0.05 or P < 0.01); the PIR group showed significantly decreased corner dwell time and significantly increased edge dwell time (P < 0.01); the VAPH and VAPL groups showed decreased corner dwell time and increased edge dwell time, but the differences were not statistically significant.

[0016] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A research method for studying the mechanism of action of deer antler peptides based on the Wnt / β-catenin signaling pathway on mild cognitive impairment, characterized in that, Includes the following steps: S1. Wistar rats were acclimatized for 7 days and then randomly divided into a blank control group, a model group, a positive control group, a high-dose VAP group, and a low-dose VAP group using a randomized parallel control method. Each group was administered the drug by gavage for 18 consecutive days. The blank control group and the model group were given an equal volume of distilled water. S2. Two hours after administration on day 18, the model group, positive control group and VAP group were injected intraperitoneally with scopolamine 2 mg / kg, while the blank control group was injected with an equal volume of normal saline. S3, Unilateral common carotid artery ligation: After adaptive feeding, except for the blank control group, the left common carotid artery of the remaining rats was ligated after anesthesia, and the drugs were administered 3 days after the operation. S4. The rats in the intraperitoneal injection scopolamine model were tested using the open field test to determine their resting time and movement time within 2 minutes; the rats in the unilateral common carotid artery ligation model were tested using the Morris water maze to determine their escape latency and number of times they crossed platforms within 3 minutes. S5. After the behavioral experiment, the rats were anesthetized and blood was collected. They were then sacrificed by cervical dislocation and the hippocampal tissue was separated. S6 and HE staining were used to observe pathological changes in the hippocampus; serum SOD, MDA, and BDNF levels were detected by ELISA; and the expression of PI3K, AKT, and Bad-related proteins in the hippocampus was detected by Western blot. S7. Using SPSS 21.0 software, express the data as mean ± standard deviation, and use one-way ANOVA to assess differences. P < 0.05 or P < 0.01 is considered statistically significant.