Effects of gastrodin on the expression of brain aging‐related genes in SAM/P‐8 mice based on network pharmacology

Abstract Background Gastrodin can reduce neuronal damage through multiple targets and pathways, and can be useful in preventing and treating degenerative lesions of the central nervous system, but the specific mechanism has not been elucidated. Methods The aging‐related genes in the hippocampus and the frontal cortex were detected in adult and aged mice treated with gastrodin or not. In addition, we collected the target genes of gastrodin and aging from a network database, and a Venn diagram was created to obtain the intersection target genes of gastrodin and aging. Then, the String database was used to analyze the protein–protein interactions (PPIs) between aging‐related genes and the target genes of gastrodin and aging. The “drug–disease–target–pathway” network was constructed using Cytoscape 3.7.2 software, and the main mechanism and pathway of key genes were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). Finally, the reliability of these key genes was further verified by molecular docking technology. Results The results showed that 6 out of 10 genes related to brain aging were differentially expressed after gastrodin intervention. Moreover, there were 11 key genes between gastrodin and differentially expressed genes related to brain aging. GO and KEGG results suggested that material metabolism and carbohydrate digestion and absorption were associated with the pathological mechanism of gastrodin antiaging. Molecular docking results also confirmed the good binding activity of gastrodin to the key genes. Conclusion Gastrodin plays a potential role in antiaging by regulating substance metabolism and carbohydrate digestion and absorption.

Aging refers to an irreversible phenomenon wherein, when organisms mature, with age, their body' function, ability to stabilize the internal environment, and ability to stress declines; this is also accompanied by degenerative changes in multiple organs and tissue structures, leading to death. 1 Current studies have shown that brain aging plays a dominant role in the aging process of the body, and the lesion sites are mainly concentrated in the hippocampus and the frontal cortex. 2,3Among these, the hippocampus is one of the most vulnerable brain regions.Diseases such as Alzheimer's disease (AD), which are closely related to learning and memory, are directly related to the functional decline of the hippocampus and are accompanied by changes in specific gene expression. 4,5ith the aging of populations worldwide and the increasing incidence of aging-related diseases, the social and economic burden is also increasing. 6At present, antiaging treatment mainly includes drug therapy, stem cell and microbial transplantation, hormone replacement therapy, and so forth.Unfortunately, the current treatment for AD has not shown significant clinical efficacy. 7n recent years, new antiaging strategies have led to new directions and hopes for treatment, but many difficulties still need to be overcome to achieve clinical application and treatment.Therefore, active exploration of the pathogenesis of aging, development of effective drugs to delay aging, and reduction of the incidence of agingrelated diseases are of great significance to improve people's living standards and quality of life.
Aging has remained an unresolved issue in the medical field.It is a multifactorial process that leads to a gradual decline in the function of cells, tissues, and organisms.Although aging cannot be stopped, it is possible to slow down the rate of aging through various methods.][10][11][12] Studies have shown that TCM can improve the neurocholine function of the body, protect cerebral vessels, and significantly improve the cognitive function of patients by acting on multiple tissues and multiple targets. 13,14Gastrodin is a phenolic glycoside isolated from the rhizome of Gastrodia elata and its tuber has been used in TCM for centuries.Its active ingredients have physiological and healthpromoting features, including antitumor, memory improvement, and neuroprotective activities. 15,168][19] Recent studies have found that gastrodin can alleviate neuronal damage due to various causes by inhibiting intracellular calcium overload, inhibiting neuronal apoptosis, stabilizing neuron membrane, and antioxidation.][22][23] Although gastrodin has shown great potential in the treatment of NDs, there are no in-depth and specific studies on whether gastrodin can improve aging by regulating antiaging genes.
With the development of interdisciplinary fields such as bioinformatics, system biology, and comprehensive pharmacology, the research strategy for exploring the interaction between drugs and diseases has gradually shifted from independent research to systematic overall analysis. 24Network pharmacology based on the concept of "drug-disease-target-pathway" measures the regulation of drugs on biological molecular networks from a systematic and holistic perspective.Its basic principle coincides with the characteristics of multiple targets and multiple functions in the treatment of diseases by TCM. 25 TCM network pharmacology, which aims to understand the network-based biological basis of complex diseases, TCM syndromes, and herb treatments, plays a critical role in the origin and development process of network pharmacology. 26Therefore, the application of network pharmacology based on systematic bioinformatics has unique advantages in studying the molecular mechanism of TCM compounds. 27onsequently, the use of new analysis methods to explore the key genes and mechanisms of antiaging of TCM will provide a new avenue for subsequent research and application of TCM.In this study, we first explored whether gastrodin affected the expression of agingrelated genes through animal experiments and then further explored the antiaging targets and possible mechanisms of gastrodin in combination with network pharmacology.At the same time, we also constructed a potential "drug-disease-target-pathway" network of gastrodin and identified the key genes by molecular docking technology to provide a theoretical and experimental basis for subsequent research (Figure 1).

| Animal grouping and treatment
The animal experimental design in this study was approved by the Animal Experimental Ethics Review Committee of Kunming Medical University (approval no.kmmu20221547).SAM-P/8 mice were purchased from the Experimental Animal Center of Tianjin University of TCM.The adult mice were 7 months old and the elderly mice were 13 months old, with a weight of (25 ± 2) g.The mice were kept in a room with the temperature maintained at 22 ± 2.0°C and humidity maintained at 50 ± 5%, with light exposure for about 12 h a day and free access to food and water.Twenty SAM-P/8 mice were randomly divided into four groups (n = 5/group): adult control group, adult gastrodin group, aged control group, and aged gastrodin group.The gastrodin injection (H20013046; Kunming Pharmaceutical Group) was diluted with medical 0.9% normal saline at 5 mg/ml, and mice in the adult gastrodin group and the aged gastrodin group were administered an intraperitoneal injection at a dose of 1 g/kg, once a day, for 14 consecutive days, with the left and right abdominal cavity alternated every other day.Neither the adult control group nor the aged control group received any treatment.

| Tissue harvest
After 14 days of feeding, mice in each group were intraperitoneally injected with 1% pentobarbital sodium 30 mg/kg.The disappearance of the tail clamp reflex indicated that anesthesia was effective.Perfusion was performed with precooled saline and a 4% paraformaldehyde phosphate buffer solution (pH = 7.2) after left ventricular catheterization.The mice were intraperitoneally injected with 1% pentobarbital sodium at 50 mg/kg.After the tail pinch reflex disappeared, the anesthesia took effect, and then the perfusion was performed.Hippocampus and frontal cortex tissues were rapidly separated and rinsed with precooled diethyl pyrocarbonate-treated water, placed into a 1.5 ml Eppendorf tube containing 1 ml of TRIzol lysis RNAase-free, and homogenized in an ice bath.RNA was isolated according to the instructions of the kit.The concentration and purity of RNA were detected by agarose gel electrophoresis and an ND-1000 spectrophotometer (UV-250IPC Shimadzu).Total RNA was isolated by TRIzol reagent (Takara Bio Inc.) and complementary DNA (cDNA) was synthesized according to the RevertAidTM First Strand cDNA Synthesis Kit (Thermo Scientific).The first strand of synthesized cDNA can be directly used for PCR amplification.According to the instructions of the 2× PCR Master Mix Kit (#K0171; Fermentas), β-actin and 10 agingrelated genes were amplified, and the primer sequences are shown in Table 1.The total volume of each reaction was 20 μl: 10 μl of 2× PCR Master Mix, 8 μl of PCR water nuclease-free, 0.5 μl of upstream primer, 0.5 μl of downstream primer, 1 μl of RT cDNA template.Thermal cycle parameters were as follows: 94°C denaturation for 5 min, 94°C denaturation for 1 min, annealing for 1 min, 72°C extension for 1 min, and 35 cycles after 72°C extension for 10 min.PCR products were detected by 1% agarose gel electrophoresis, and gel images were taken under the ultraviolet mode of the BIO-GEL gel imager to determine the gray value of each target gene band (WD-9403c; Beijing Liuyi Instrument Factory).Quantity One software was used to analyze electrophoresis bands and calculate the ratio of target gene bands to the β-actin gray value.

| Screening of gastrodin and aging targets
The chemical structure of gastrodin was obtained from the chemical information database PubChem using "Gastrodin" as the keyword and its "Canonical SMILES" sequence was found (https://pubchem.ncbi.nlm.nih.gov/).Swiss Target Prediction (http://swisstargetprediction.ch/) is a website that can predict possible targets according to the chemical structure of drug components.The canonical SMILES sequence of gastrodin was imported into the website for target prediction.In addition, the collected targets were converted into target names and gene names in the Uniport database (https://www.uniprot.org/).With "aging" as the keyword, all relevant disease targets were found in the GeneCards database (https://www.genecards.org/) and downloaded for analysis.After taking two medians of correlation, targets with correlations >1.43 were identified.

| Core target analysis and protein-protein interaction (PPI) network construction
The screened gastrodin and aging-related targets were uploaded to the plotting website Jvenn (http://www.bioinformatics.com.cn/static/others/jvenn/example.html), and the Venny diagram was plotted to find the common targets of drugs and diseases.These common targets were the required core targets.String (https://string-db.org/) is a website that can analyze the relationship between proteins online.The String database was used to input 11 crosslinked targets of gastrodin and aging together with six changed targets in the experimental results into the String to search and continue to produce protein interaction maps.The high-definition interaction diagram and the interaction table were exported and downloaded.The interaction table was imported into Cytoscape 3.7.2.According to the degree value and combined_score, nodes with different sizes and connection lines with different thicknesses were generated to draw a PPI network diagram.

| Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis
In this study, the common targets of gastrodin and aging were analyzed by bioconcentration, and the genes and gene products were annotated by GO analysis according to the biological process (BP), the cellular component (CC), and the molecular function (MF).KEGG is a useful resource for the systematic analysis of gene function and related highlevel genomic function information.Therefore, to further clarify the function of the selected intersection genes and their roles in the signal transduction pathway, GO function analysis and KEGG pathway enrichment analysis were performed using the DAVID database (http://david.abcc.ncifcrf.gov/),and the p value and fold enrichment value were determined.

| Molecular docking
Molecular docking has become an important technology for computer-aided drug research in recent years.It can be used to evaluate the interaction between active ingredients and core targets and predict their binding modes and affinity.The three-dimensional structure file of the core protein was obtained from the RCSB protein database (http://www.rcsb.org/).The crystal structure of the protein was initialized using AutoDockTools software (version: 1.5.6),including removal of solvent molecules and ligands, addition of polar

| Statistical analysis
All data in this study were processed using SPSS 21.0 statistical software, and all measurement data were expressed as mean ± standard deviation (x ¯± SD).The ratio of the average optical density of each gene β-actin between the two groups was tested using an independent-sample t-test, and p < 0.05 indicated that the difference was statistically significant.

| Effects of gastrodin on aging-related genes
The expression of β-actin and aging-related genes was detected in the hippocampus and the frontal cortex of all groups, and the target gene fragment was obtained, which was consistent with the expected fragment length (Figures 2 and 3).Grayscale scanning was performed on the image, and the ratio of the image to β-actin was calculated after data correction for statistical analysis.The results showed that these 10 genes were expressed in the hippocampus and the frontal cortex of SAM-P/8 mice treated with gastrodin, although the levels were different.These 10 genes may be involved in maintaining the physiological functions of the hippocampus and the frontal cortex, and the maintenance of their expression may be necessary in the aging process of the hippocampus and the frontal cortex in mice, but the specific functions are different.We compared the adult gastrodin group with the adult control group and the aged gastrodin group with the aged control group.The results are shown in Figure 4. Compared with the control group, the expression of SCN2B was increased and the expression of Sortilin-1 was decreased in the hippocampus and the frontal cortex of adult mice treated with gastrodin.The expression of MAP1B and RAP2A increased in the frontal cortex (Figure 4, p < 0.05).
Compared with the control group, the expression of MAP2K4, Sortilin-1, and Rab6A in the hippocampus and the frontal cortex of aged mice decreased (Figure 4, p < 0.05).

| Acquisition of the intersectional core target genes of gastrodin and aging
Through database retrieval, 16 target genes of gastrodin were screened (Figure 5A).There were more than 20,000 target genes related to aging.After taking two medians of correlation, 6670 target genes with a correlation >1.43 were determined.The Venny map was created using an online drawing website, and 11 core target genes of gastrodin and aging were obtained, including TYR, CDA, ADORA2A, SLC5A2, ADORA1, AKR1B3, SLC5A1, FUCA1, MGAM, FOLH1, and ADA (Figure 5B).

| PPI network of target genes
The PPI network can be used to discover multiple interactions among multiple proteins in diseases and determine potential therapeutic targets. 28The interaction between 11 selected genes and 6 changed genes in the experimental results was determined using the String database to determine the mode of interaction and the score between nodes, and the PPI network was constructed.Cytoscape 3.7.2 was used to set up the connection lines with different sizes and different thicknesses according to the degree value and combined_score, and the PPI network diagram was constructed.Green represents the gene mentioned in the experimental results and orange represents the gene of the core target.Furthermore, the size of each point was proportional to its participation in the network (Figure 5C).The results showed that the main core genes in the network included Mgam, Rab6a, and Ada, which were mainly involved in starch digestion and hydrolysis, protein transport regulation, and purine metabolism.

| Bioinformatics analysis
To explore the biological function of core targets, GO function and KEGG pathway enrichment analyses were performed.Using the DAVID database, GO enrichment analysis of 17 target genes involved in the PPI network was carried out, and 42 enrichment results were obtained, including 19 BP items, 17 CC items, and 6 MF items (Figure 5D).Similarly, the KEGG pathway analysis of key gene genes showed that the antiaging mechanism of gastrodin was mainly related to the three pathways of galactose metabolism, metabolic pathways, and carbohydrate digestion and absorption (Figure 5E).The 17 key targets and pathways of gastrodin were mapped to the "target-pathway" network by Cytoscape 3.7.2software.To establish the network more conveniently, the pathway of gastrodin is represented by hsaID.Based on the network analysis, the network topology characteristics of the network model are analyzed (Figure 5F).The results showed that the core pathways, including galactose metabolism, metabolic pathways, and carbohydrate digestion and absorption, were the potential therapeutic processes of gastrodin in delaying aging.Seventeen genes are interrelated, which may be the key target genes for gastrodin to delay aging.

| Molecular docking of key genes
In this study, the possible interactions between 17 key target proteins and gastrodin in the experimental and network pharmacology analysis results were verified by molecular docking.AutoDock Vina software was used to perform molecular docking between gastrodin and 17 proteins such as SCN2B (Figure 6).The docking results were expressed as the affinity score.The smaller the value, the stronger the binding force.When affinity ≤ −7 kcal/mol, it showed strong binding activity. 14In this experiment, MAP1B, TYR, SLC5A2, and ADORA1 had no protein structure docking.Among the 13 docking proteins, the docking scores were less than −7 kcal/mol, except for SORT1, MGAM, and ADA proteins, indicating that gastrodin may have good binding activity with these proteins.The above results suggest that the expression of these proteins may play an important role in the antiaging of gastrodin (Table 2).

| DISCUSSION
In this paper, the molecular mechanism of gastrodin antiaging was explored by combining experimental verification using mice and network pharmacology analysis.The experimental results obtained from mice showed that gastrodin could regulate brain aging-related genes, which might act on target proteins SCN2B, Sortilin-1, MAP1B, RAP2A, MAP2K4, and Rab6A.To further explore the molecular mechanism by which gastrodin improves aging, the potential targets and mechanism of gastrodin antiaging were analyzed by screening, target prediction, and bioinformatics analysis using network pharmacology and a public database.
From the results of network pharmacology analysis, 11 gastrodin and aging intersection genes were constructed with six genes changed in the experiment to construct PPI, and GO and KEGG analyses were performed to explore the three most relevant pathways.The "drug-disease-target-pathway" diagram was drawn and verified by molecular docking technology.The results showed that 10 of the 17 proteins had high binding activity to gastrodin.SCN2B can affect the formation and transmission of neuronal action potential by regulating the sodium ion channels, which plays an important role in the maintenance of neuronal physiological functions, and is related to aging and memory decline. 29,30 | 165 gene, is a key molecule regulating neuronal activity and function. 31It not only contributes to the functional integrity of the nervous system under physiological conditions but also plays an important role in neuronal injury and disease. 32MAP1B is the first microtubuleassociated protein expressed during the development of the nervous system, which can play an important role in nerve development and neuronal differentiation by regulating microtubule dynamics and promoting axon growth and development. 33ADORA2A and ADORA1 are subtypes of adenosine receptors, widely distributed in various tissues and organs of the human body, highly expressed in the central system, and involved in mediating neuronal development, sleep and awakening, learning and memory, motor function and drug addiction, and a series of important processes. 34SLC5A1 and SLC5A2 are solute carrier family transporters, which are one of the most important membrane transporters on the human cell membrane.They play a role in the development of neuropsychiatric disorders by changing the expression, dysfunction, and regulation of neurotransmitter transporters. 35,36AKR1B3 is a detoxification enzyme of TCM, which not only participates in plasma glucose energy metabolism and reactive oxygen species production but also in the proliferation of astrocytes through the Akt pathway to promote the repair of nerve damage. 37It is worth noting that there are some genes in our analysis results that have not been reported in relation to aging and the nervous system before, indicating that we can further explore the function of these genes in future experimental studies.
Given the current knowledge of these core targets, it can be inferred that gastrodin may play an antiaging role by promoting neurological development, regulating neuronal activity and function, altering the expression of neurotransmitter transporters, and participating in the proliferation of astrocytes to promote the repair of neural injury.Meanwhile, the results of GO enrichment analysis of key targets indicated that the response to substances, regulation of the sleep cycle, glucose transport and absorption, and regulation of the adenosine receptor signaling pathway play an important role in the antiaging of gastrodin.KEGG pathways enriched by these key genes mainly include galactose metabolism, metabolic pathways, and carbohydrate digestion and absorption, suggesting that gastrodin may play an antiaging role by regulating substance metabolism and carbohydrate digestion and absorption.Metabolic dysfunction may play an important role in delaying aging.9][40] Metabolic interventions through calorie restriction or modulation of key metabolic signaling pathways are currently the focus of research focused on maintaining health and slowing aging. 41,42Several studies in recent years have shown that by regulating key signaling pathways in glucose and energy metabolism, the health of various organisms can be improved and lifespan can be extended. 43,44Some studies have reported that most of the current interventions to prevent and delay aging are achieved by targeting central metabolic pathways. 45In general agreement with previous studies, the results of our experiment have shown differences in the expression of aging-related genes in the brain after gastrodin intervention.The results of network pharmacology analysis indicated that gastrodin may exert its antiaging effects by regulating signaling pathways related to substance metabolism and carbohydrate digestion and absorption.Although we have explored the targets and mechanisms of the antiaging effects of gastrodin through network pharmacology and molecular docking techniques, the underlying mechanisms are still unclear.The main limitation of this study is that no experimental validation of the screened key targets and possible mechanisms has been conducted, and it has not been determined whether gastrodin has the effect of regulating aging-related genes.What are its antiaging targets and mechanisms of action?We will conduct further experimental studies to verify this in the follow-up study.In summary, in this study, aging-related genes were detected in the hippocampus and the frontal lobe of aging mice by RT-PCR, and the data were analyzed in combination with network pharmacology.According to the results of analyses, it is speculated that the antiaging effect of gastrodin may be related to the regulation of material metabolism and carbohydrate digestion and absorption, which provides an avenue for further verification of its mechanism in future experimental studies.

F
I G U R E 1 Flowchart of the study design [Color figure can be viewed at wileyonlinelibrary.com] chain reaction (RT-PCR) T A B L E 1 Gene primer sequence, renaturation temperature, and product length initialization, and so forth, and converted into the pdbqt format file required for docking.The threedimensional structure of gastrodin was optimized by the MM2 force field using Chem3D Pro 14.0 software (version: 14.0.0.117), and the number of rotatable keys was set by AutoDockTools software and converted into the pdbqt format file required for docking.Molecular docking scoring was performed using the molecular docking software AutoDock Vina (version: 1.1.2).

F I G U R E 3
One percent agarose gel electrophoresis of polymerase chain reaction products of β-actin and aging-related genes in the frontal cortex.(A) β-Actin, (B) Calcineurin-B, (C) Calmodulin-1, (D) MAP1B, (E) MAP2, (F) MAP2K4, (G) Rab6A, (H) RAP2A, (I) Regucalcin, (J) SCN2B, (K) Sortilin-1.The swimming lanes from left to right are the DL2000 marker (2000, 1000, 750, 500, 250, and 100), the adult control group, the adult gastrodin group, the aged control group, and the aged gastrodin group.F I G U R E 4 Expression of aging-related genes in SAM-P/8 mice.(A) Gene expression in the hippocampus of the adult control group and the gastrodin group.(B) Gene expression in the frontal cortex of the adult control group and the gastrodin group.(C) Gene expression in the hippocampus of the aged control group and the gastrodin group.(D) Gene expression in the frontal cortex of the aged control group and the gastrodin group.*p < 0.05.[Color figure can be viewed at wileyonlinelibrary.com] Sortilin-1, a transmembrane neuropeptide receptor encoded by the SORT1 F I G U R E 5 Screening of key targets and biological function analysis.(A) Core targets of gastrodin.(B) Wayne plot of the intersection target of gastrodin and aging.Green and blue circles represent the screened gastrodin and aging targets, respectively, and the middle part represents the common target of gastrodin and aging.(C) Interaction map of intersection targets and experimental targets.The larger the node area, the more important it is in the network.(D) Gene Ontology enrichment analysis of key genes of gastrodin antiaging.(E) Enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes pathway of gastrodin antiaging key genes.(F) "Target-pathway" network diagram of gastrodin antiaging.[Color figure can be viewed at wileyonlinelibrary.com]ZHAO ET AL.

F I G U R E 6
Molecular docking diagram of gastrodin and key targets [Color figure can be viewed at wileyonlinelibrary.com] T A B L E 2 Molecular docking information of key targets