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Original Article

Korean J Physiol Pharmacol 2024; 28(4): 361-377

Published online July 1, 2024 https://doi.org/10.4196/kjpp.2024.28.4.361

Copyright © Korean J Physiol Pharmacol.

Analysis of the mechanism of fibrauretine alleviating Alzheimer's disease based on transcriptomics and proteomics

Lu Han1, Weijia Chen1, Ying Zong1, Yan Zhao1, Jianming Li1, Zhongmei He1,2,3,*, and Rui Du1,2,3,*

1College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun 130118, 2Key Laboratory of Animal Production, Product Quality and Security, Ministry of Education of China, Changchun 130118, 3Jilin Provincial Engineering Research Center for Efficient Breeding and Product Development of Sika Deer of China, Changchun 130118, China

Correspondence to:Zhongmei He
E-mail: 007014@jlau.edu.cn
Rui Du
E-mail: durui@jlau.edu.cn

Received: November 12, 2023; Revised: January 17, 2024; Accepted: January 18, 2024

The dried rattan stem of the Fibraurea Recisa Pierre plant contains the active ingredient known as fibrauretine (FN). Although it greatly affects Alzheimer's disease (AD), the mechanism of their effects still remains unclear. Proteomics and transcriptomics analysis methods were used in this study to determine the mechanism of FN in the treatment of AD. AD model is used through bilateral hippocampal injection of Aβ1-40. After successful modeling, FN was given for 30 days. The results showed that FN could improve the cognitive dysfunction of AD model rats, reduce the expression of Aβ and P-Tau, increase the content of acetylcholine and reduce the activity of acetylcholinesterase. The Kyoto Encyclopedia of Genes and Genomes enriched differentially expressed genes and proteins are involved in signaling pathways including metabolic pathway, AD, pathway in cancer, PI3K-AKT signaling pathway, and cAMP signaling pathway. Transcriptomics and proteomics sequencing resulted in 19 differentially expressed genes and proteins. Finally, in contrast to the model group, after FN treatment, the protein expressions and genes associated with the PI3K-AKT pathway were significantly improved in RT-qPCR and Western blot and assays. This is consistent with the findings of transcriptomic and proteomic analyses. Our study found that, FN may improve some symptoms of AD model rats through PI3K-AKT signaling pathway.

Keywords: Alzheimer's disease, Fibrauretine, Proteomics, Transcriptomics

One of the most prevalent neurodegenerative illnesses with dementia-like features, Alzheimer’s disease (AD) has a complex pathogenesis and numerous influencing factors [1]. It is characterized by severe neuronal deficits, extracellular amyloid β (Aβ) plaques, as well as intracellular Tau hyperphosphorylated neurofibrillary tangles (NFT) [2,3], of which Aβ accumulation has a critical part in AD’s pathogenesis. As a result, inhibition of Aβ deposition to alleviate the onset of AD-related symptoms has become a popular topic of research. A wide range of medical uses for fibrauretine (FN), an isoquinoline alkaloid isolated from the dried rattan stem of Fibraurea Recisa Pierre, includes the treatment of bacterial infections, anti-inflammatory, anti-diabetic, antioxidant, immune enhancement, and improvement of a number of diseases [4]. Our research has demonstrated significant improvement of AD by FN [5]. Thus, in order to evaluate the mechanism of action of FN on AD, our study established an animal model of the disease via Aβ induction.

Recent advancements in large-scale high-throughput sequencing technologies have made it possible to gain a more comprehensive understanding of the pathological processes underlying diseases through the use of histological analysis. Among them, both transcriptomics and proteomics have been extensively utilized to find therapeutic targets for diseases. A large proportion of differentially expressed transcription factors and proteins has been found to be functionally related to the molecular biology of learning and memory [6]. This enables a global perspective to display the protein expression and transcriptional levels of all genes in an organism at a given time, to better study pathogenesis and screen for biomarker indicators. Proteomics validation analysis has shown that the deposition of Aβ triggers microglia proteome alterations and dysfunction in a mouse model of AD, revealing protein “signature” changes in various stages of AD. So far, the exploration of the pathogenesis of AD has not been systematically investigated in proteomics and transcriptomics, and more and more effective therapeutic approaches are yet to be elucidated. Hence, in order to create a foundation for the clinical prevention and treatment of AD, we start independently from a genetic and protein viewpoint, investigation by transcriptomics and proteomics, and further exploration of the FN mechanism of action in AD therapy and prevention.

Animals and reagents

Fibrauretine (Yunnan Plant Pharmaceutical Co., Ltd., State Drug Quotient: Z53020154, Lot No.: 1407067), Protein lysis buffer (7M urea, 2M thiourea, 0.1% CHAPS), Urea (Bio-Rad, Lot No.: 161-0731, USA), Thiourea (Sigma-Aldrich, Lot No.: T7875, USA), CHAPS (Bio-Rad, Lot No.: 161-0460, USA), Protease inhibitor (Shanghai Limin Industrial Co., Ltd., model: 04693132001/kit), Ultrasonic cell disruptor (Nanjing Xianou Instrument Manufacturing Co., Ltd., model: XO), Orbitrap Fusion (Thermo, model: Orbitrap Fusion), Aβ1-40 (MedChemExpress, Lot No.: 131438-79-4), sodium carboxymethylcellulose (Shanghai Yuanye Biotechnology Co., Ltd., Lot No.: 9004-32-4), SD rats (male, weight 190–220 g, SPF grade). All rats were provided by YISI Experimental Animals Co., Ltd. (license No.SCXK (Ji)-2020-0002). All animals were acclimatized and fed at a humidity of 60% ± 10% and room temperature of 23°C ± 2°C for 1 week, and all rats were allowed to drink and eat freely during the experimental period [7]. The Animal Care and Use Committee of the Academy of Chinese Medical Sciences approved these animal studies (Animal Ethics No: 2021 07 26001), which were then carried out in accordance with the “Guide for the Care and Use of Laboratory Animals” [8].

AD model construction and drug administration

All rats are randomly created in three groups: Control, Model and FN. Each group had 10 rats. All rats were anesthetized with 1% pentobarbital sodium (intraperitoneal injections, 40 mg/kg). On the brain stereotaxic apparatus, they were fixed, a small amount of roxithromycin ointment was applied to both eyes, the hair on the head was shaved off with an animal-specific shaver, and alcohol cotton balls were sterilized. Moreover, a scalpel and scissors were used to make a longitudinal incision in the center of the skull and the fascia as well as the subcutaneous tissue were separated to reveal the skull. In addition, in order to stop bleeding, sterile dry cotton was utilized, and marks were made in the anterior chamber. With the fontanelle serving as a reference point, the CA1 region of the hippocampus was employed as the drug delivery point bilaterally. 2.3 mm to the left and right of the sagittal line, and 3.5 mm to the back, a hole about 0.6 mm in size was drilled on the skull surface with a micro cranial drill. In the model group and FN groups, 1 µl of Aβ1-40 (5 µg/µl) solution was injected with a microinjection needle at a constant rate of 0.2 µl/min. The injection depth was 3.5 mm, and the needle was in situ for 5 min, after which the needle was slowly pulled out. The control groups was injected with 1 µl 10% normal saline instead of Aβ1–40. After the needle is removed, the wound is sutured. The operation was the same, and penicillin was injected for 3 consecutive days following surgery to avoid infection. Three days after surgery, FN group was intragastrically administered FN (p.o, 80 mg/kg) for 30 days. The control group and the model group were given an equivoluminal physiological saline containing 0.05% carboxymethylcellulose sodium for 30 days. Simple experimental groups such as Table 1.

Table 1 . Simple experimental grouping.

GroupModeling drugsTreatments
Control10% normal saline 1 μl10% normal saline 30 days
Model1–40 5 ug/ul 1 μl10% normal saline 30 days
FN1–40 5 ug/ul 1 μlFN 80 mg/kg 30 days

FN, fibrauretine; Aβ, amyloid β.



Preparation of samples

All rats were decapitated and blood was centrifuged for 15 min at 12,000 r/min so as to collect the supernatant for backup, and the brain tissue was removed on ice, and part of it was fixed in 4% paraformaldehyde, the other part was kept in –80°C refrigerator for backup.

Morris water maze test

Morris water maze test is a common test method of learning and memory. Morris water maze consists of a circular pool with a diameter of 1,200 mm, filled with water. The pool is divided into four according to the positioning mark. After 30 days of FN treatment, a 5-day Morris water maze test was performed to evaluate the learning and memory ability of rats. The positioning navigation experiment was carried out 4 days before the experiment. Put the rat into the pool from the fourth quadrant and record the escape latency, the maximum latency was 120 sec. If the rat did not find the platform within 120 sec, the rat was guided to find the platform. On the fifth day, the space exploration experiment was carried out. After removing the platform, the rats were put into the pool from any quadrant, record the number of times the rat passes through the target area [9].

Enzyme linked immunosorbent assay (ELISA) of acetylcholine (Ach) and acetylcholinesterase (AChE) activity measurement

According to the instructions of the manufacturer, the activity of AChE and ACh in serum was measured. Furthermore, the absorbance values were detected and calculated at 405 nm with an enzyme marker for the purpose of observing the changes in choline function in each group.

Immunohistochemical detection of hippocampal Aβ and P-Tau protein

Immunohistochemical analysis was carried out for detecting the Aβ and P-Tau protein expression levels in brain tissues. Fresh hippocampal tissues from each group of mice were taken, which were dehydrated in ethanol, transparent in xylene, embedded in paraffin, sectioned (continuous sagittal plane of brain, 30 µm), sealed with 3% hydrogen peroxide for 15 min for endogenous peroxidase and serum for 15 min for non-specific antigen, incubated with primary antibody P-Tau at room temperature (1:100–500) for 1 h and then overnight at 4°C. The smear was removed the following day, cleaned three times with PBS, incubated for 1 h with a secondary antibody (1:500), color developed with diaminobenzidine for the 30 sec, rinsed and re-stained with hematoxylin. After differentiation, dehydration, and transparency, the smear was sealed with a drop of gum and observed under an inverted microscope.

RNA-Seq of rat brain tissue

Extraction and identification of total RNA: NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific) was utilized to determine the RNA’s purity and concentration, and Agilent Bioanalyzer 2100 system (Agilent Technologies) was employed to evaluate the integrity of the RNA. In order to make sure that the samples were of acceptable quality for library sequencing analysis, the RNA integrity was checked using the RNA Nano 6000 test kit. Three biological replicates of each set were performed.

Construction of mRNA libraries: Each sample weighed a total of 1 µg, which was utilized to construct the initial library. Utilizing magnetic beads containing oligo (DT) as directed by the manufacturer, mRNA was extracted from total RNA, mRNA was fragmented by random interruption and the first cDNA strand was produced utilizing the SuperScript double-stranded cDNA synthesis kit (Invitrogen) employing mRNA as the template. The DNA fragment is then adenylated at the 3' end and attached to a NEBNext junction by a hairpin loop structure. The nucleic acid purification kit AMPure XP magnetic beads (Beckman Coulter) were used to purify the library fragments. After that, 3 µl of USER Enzyme (NEB) was incorporated, incubated for 15 min at 37°C and then reacted for 5 min at 95°C before PCR. Then, using universal PCR primers, index (X) primers, and high-fidelity DNA polymerase, PCR was carried out. After PCR products were purified. Agilent Bioanalyzer 2100 equipment was used to evaluate the quality of the library.

Library quality control and sequencing: Initial quantification was conducted following library construction via Qubit 3.0 fluorescence quantification instrument at a concentration of 1 ng/ul or more. The library is then accurately quantified using Q-PCR to guarantee library quality. The libraries were sequenced in PE150 mode utilizing the Illumina NovaSeq6000 sequencing platform in accordance with the guidelines of the manufacturer after passing quality control.

Expression and enrichment analysis of differential genes: After sequencing data was off-boarded, the resulting data was examined via the bioinformatics analysis procedure offered by the BMKCloud (www.biocloud.net), a Bemac cloud platform. The data were filtered for obtaining Clean Data, and the Hisat2 (version 2.2.1, --dta-p 6--max-intronlen 5000000) tool was used to perform sequence comparison with the specified reference genome. Samples were analyzed using edgeR analysis (version 3.32.1, dispersion = 0.16), with fold change (FC) ≥ 1.5 and false discovery rate (FDR) < 0.05 set as the threshold for significant differential expression. The screened differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis, which in turn identified the signaling and metabolic pathways in which the differentially expressed genes were mostly engaged. Utilizing the STRING database (http://string-db.org/), the protein-protein interaction network maps of differentially expressed genes were created, and Cytoscape 3.7.2 was used to visualize the reciprocal networks.

After transcriptome sequencing analysis, Nr4a1, SPP1 and KLF4 were selected for RT-qPCR verification.

Quantitative real-time PCR (qPCR) analysis: The brain tissue was removed and evenly crushed, and total RNA was extracted based on Trizol instructions [10,11]. The products were obtained after reverse transcription in accordance with the operating instructions of the reverse transcription kit, and then the reaction was performed according to the operating instructions of the SYBR Premix Ex TaqTM kit. The reaction conditions are as follows: initial denaturation for 3 min at 95°C, followed by 40 cycles at 95°C for 30 sec, 50°C for 30 sec, and 68°C for 90 sec. After measuring the cycle threshold values of the samples, the 2-ΔΔCT approach was adopted. CYP450 enzyme gene expression’s FC was calculated for each group relative to the control group [12].

Proteome sequencing of brain tissue from rats

Protein extraction and quality control: Brain tissues from each group were weighed 100 mg and grounded in a mortar and pestle until homogenized, followed by adding protease inhibitor, sonicated for 1 sec, stopped for 2 sec, and accumulated for 120 sec. 14,000 g was centrifuged for 20 min and the supernatant was quantified. Bradford’s technique was used to estimate the concentration of the protein. 10 µg of each group was subjected to 12% SDS-PAGE gel electrophoresis, followed by Kemas Brilliant Blue R-250 staining and decolorization until the bands were clear.

Enzymatic digestion and desalting of proteins: To the final concentration, add 10 mM dithiothreitol, and then proceed to a 1-hour incubation at 37°C before returning to room temperature. Iodoacetamide was added at a final concentration of 30 mM and left to incubate at room temperature for 45 min without exposure to light. It was then diluted 8 times with 50 mM ABC and trypsin (trypsin:protein = 1:25) was added overnight at 37°C. The next day, samples were desalted utilizing a C18 desalting column, activated with 100% acetonitrile (ACN), equilibrated with 0.1% formic acid (FA), loaded onto the column, washed with 0.1% FA to remove impurities, and then eluted with 70% ACN, lyophilized, and subjected to peptide concentration determination for liquid chromatography-mass spectrometry (LC-MS) analysis.

Nanoscale reversed-phase chromatography Orbitrap Fusion for protein analysis: Mobile phase A was prepared in 100% MS water and 0.1% FA while liquid B was prepared in 0.1% FA and 100% ACN. The components obtained by lyophilisation were re-dissolved in 0.1% FA and 20 µl of 2% methanol, centrifuged at 12,000 r/min for 10 min, and 10 µl of the supernatant sample was taken for detection. The separation flow rate is 300 nl/min, and the separation gradient is shown in the Table 2. Ion source parameters: Spray voltage: 2.2 kv, Capillary Temperature: 350°C, Ion Source: NSI, Full MS: Resolution: 120,000 FWHM, Full Scan AGC target: 4e5, Full Scan Max IT: 50 ms, Scan range: 350–1,550 m/z, dd-MS2: Resolution: 15,000 FWHM, AGC target: 5e4, Maximum IT: 50 ms, Fragmentation Methods: host cell residual DNA, normalized collision energy: 30%. The proteomics analysis was completed via Orbitrap Fusion mass spectrometry and the raw files of the generated mass spectra were processed by means of Protein Discovery 2.4 software.

Table 2 . Separation gradient.

Time (min)A: 0.1% FA/H2OB: 0.1% FA/ACN
097%3%
592%8%
10778%28%
11020%80%
12020%80%

FA, formic acid; ACN, acetonitrile.



Data analysis and processing: The RAW files obtained by LC-MS were pooled and the libraries were searched using Protein Discovery 2.4 software [13], the Sequest HT search engine was used, the retrieval parameter settings are shown in Table 3. Percolator was used to filter peptide spectral matches and peptides to a FDR of less than 5%. After spectral assignment, peptides were assembled into proteins and were further filtered based on the combined probabilities of their constituent peptides to a final FDR of 5%. Only unique were considered for quantification. The condition for screening differentially expressed proteins is FC ≥ 1.5 and FDR < 0.05. For the purpose of further clarifying the biological pathways, cellular components, molecular functions, and cellular pathways involved in the proteomic screening of the resulting proteins, all differential proteins were mapped to various terms in the GO database, the number of proteins in each term was calculated, and then hypergeometric tests were applied to identify GO terms that were significantly enriched in differential proteins compared to all protein backgrounds. Using p-value ≤ 0.05 as the threshold, the parameters are as follows: , among them, N is the number of proteins with GO annotation information among all proteins, n is the number of differentially expressed proteins in N, M is the number of proteins annotated to a GO entry among all proteins, and m is the number of differentially expressed proteins annotated to a GO entry. GO terms that meet this condition are defined as GO terms that are significantly enriched in differentially expressed proteins. KEGG and GO analysis parameters are the same. After that, carry out functional annotation and key protein interaction analysis.

Table 3 . Proteome Discoverer database search parameters.

ParametersValue
EnzymeTrypsin
Static modificationCarbamidomethyl (C)
Dynamic modificationM oxidation (15.995 Da),
N-terminal
SpeciesHUMAN
Precursor ion mass tolerance± 15 ppm
Fragment ion mass tolerance± 20 mmu
Max missed cleavages2


After proteomic sequencing analysis, the enriched representative pathways were selected and pathway-related protein expression was discovered using a Western blot assay.

Western blot analysis: A small amount of brain tissue was taken and homogenized in RIPA lysate, the supernatant was gathered, and the protein concentration was determined via a BCA protein concentration kit. The supernatant was then sampled by a microsampling needle, separated by SDS-PAGE, and transferred to the PVDF membrane. It was then incubated at room temperature for 1 h with 5 percent milk powder, closed at 4°C with primary antibody MTOR (1:500), PI3K (1:1,000) and AKT (1:1,000–1:8,000). Followed by incubating overnight, washing 3 times with Tris-Hcl, NaCl, tween20 (TBST) for 10 min each on the next day, incubating with secondary antibody for 2 h, washing three times with TBST for 10 min each. Lastly, the blots were visualized via an enhanced chemiluminescence detection kit so as to compute the relative expression of the target protein. The experimental design is shown in Fig. 1.

Figure 1. The experimental process of analyzing the mechanism of FN improving AD symptoms based on transcriptomics and proteomics. FN, fibrauretine; AD, Alzheimer’s disease.

Statistical analysis

All values were expressed as means ± standard deviation. SPSS 20.0 statistical software was used for one-way analysis of variance and t-test. The omics data were analyzed using edgeR analysis (version 3.32.1, dispersion = 0.16), with FC ≥ 1.5 and FDR < 0.05 set as the threshold for significant differential expression. ImageJ software was used for image analysis and GraphPad Prism 8 was used for plotting. p < 0.05 was deemed significant statistically.

Effects on learning and memory of AD mice

In order to better verify whether the AD model mice were successfully established, it was found by Morris water maze experiment. In positioning navigation experiment, compared with the control group, the escape latency of the model group was significantly prolonged, and the length of the swimming path to the platform was significantly increased, after FN treatment, there was a significant improvement (Fig. 2A). In the space exploration experiment, compared with the control group, the number of rats crossing the platform position in the model group was significantly reduced. After treatment with FN, the number of crossings of rats across the platform position was increased (Fig. 2B). Fig. 2C and D shows the thermal infrared trajectories of each group of rats in space exploration experiment and positioning navigation experiment.

Figure 2. Result of Morris water maze experiment. (A) Escape latency. (B) Time of crossing platform. (C) Thermal infrared trajectories of positioning navigation. (D) Thermal infrared trajectories of space exploration. Values are presented as mean ± SD. N = 10. FN, fibrauretine. ##p < 0.01 vs. control group. **p < 0.01, *p < 0.05 vs. model group.

The effect of FN on ACh content and AChE activity in rat brain tissue

To evaluate the effect of FN on the brain of AD rats and verify whether FN can treat AD by improving the remission of choline dysfunction, we tested the activity of AChE and ACh, as shown in Table 4, we found that the ACh content in the brain of the model group was significantly lower than that of the control group (p < 0.01), and AChE activity was considerably higher (p < 0.01), whereas the ACh content in the FN group was substantially lower (p < 0.01), and AChE activity was considerably decreased (p < 0.01) than that of the model group.

Table 4 . Effect of FN on ACh content and AChE activity in rat brain tissue.

GroupACh (ug/ml)AChE (U/ml)
Control42.4138 ± 3.3627.56718 ± 0.99756
Model22.2414 ± 2.069##14.4599 ± 0.9638##
FN32.2931 ± 2.8104*8.78692 ± 1.06748**

Values are presented as mean ± SD. N = 6. FN, fibrauretine; Ach, acetylcholine; AChE, acetylcholinesterase. ##p < 0.01 vs. control group. **p < 0.01, *p < 0.05 vs. model group.



The FN effect on Aβ and P-Tau protein expression in rat hippocampus

Aβ forms plaques and P-Tau protein forms NFT, which together endanger the health of neurons. Extensive deposition of Aβ plaques in the cerebral cortex and NFT caused by P-Tau protein lesions in the cortex, it is the most significant pathological marker of AD [14]. Both of them are also closely related to the pathogenesis of AD. In order to further verify the pathological features of AD and the therapeutic effect of FN on AD, we performed immunohistochemical detection of Aβ and P-Tau proteins. The results are shown in Fig. 3, we find the control group showed a few plaques with intact cell morphology and structure, no positive expression of Aβ, and less positive expression of P-Tau. In the model group, P-Tau and Aβ protein expression were significantly increased in contrast to the control group, with the difference being more significant than the control group. The FN group’s positive cells were noticeably less than those in the model group, demonstrating that FN had an inhibitory effect on P-Tau and Aβ proteins in the AD model rats.

Figure 3. The effects of FN on the contents of A β and P-Tau in hippocampus. (A) The expression of Aβ and P-Tau was detected by immunohistochemistry (200×). (B) Average optical density of Aβ. (C) Average optical density of P-Tau. Values are presented as mean ± SD. N = 6. FN, fibrauretine; Aβ, amyloid β. ##p < 0.01 vs. control group. **p < 0.01, vs. model group.

Differentially expressed genes analysis

Screening of differentially expressed genes: The distribution of differentially expressed genes between the different groups was displayed on a volcano plot (Fig. 4A, B). Overall 622 differentially expressed genes were acquired in the model and control groups, of which 323 genes were down-regulated and 299 genes were up-regulated. In the FN group, overall 518 significantly differentially expressed genes were acquired in contrast to the model group, of which 289 were down-regulated and 229 were up-regulated. Among the differentially expressed genes between the control group and model group, as well as between the model group and FN group, there are a total of 199 identical differentially expressed genes, as shown in Fig. 4C. Among them, 196 genes had opposite expression trends, and only 3 genes had the same expression trend. The information on common differentially expressed genes is listed in Supplementary Table 1. We randomly selected 3 disease-related genes for subsequent RT-qPCR validation. In order to visually display the expression differences of genes in different groups and explore new functional genes, hierarchical clustering analysis was conducted on all differentially expressed genes screened. Genes with the same or similar expression patterns in different samples were clustered and displayed through heat maps (Fig. 4D).

Figure 4. Differentially expressed genes between groups. (A) Volcano plot of differentially expressed genes between the control and model group. (B) Volcano plot of differentially expressed genes between the model and FN group. (C) Venn of common differentially expressed genes in the control group and model group, model group and FN group. (D) Differentially expressed genes clustering heatmap. N = 3. FN, fibrauretine.

GO enrichment analysis of differentially expressed genes: Comparing the brain tissues of the model and control groups, during the biological process, differentially expressed genes were primarily enriched in negative regulation of penile erection, commitment of neuronal cells to specific neuron type on forebrain and cerebrospinal fluid secretion (Fig. 5A). Among the cellular components, differentially expressed genes were mostly enriched in the transcription factor AP-1 complex, dynein complex (Fig. 5B). Among the molecular functions, differentially expressed genes were mainly engaged in transcriptional activator activity, oxygen binding, RNA polymerase II transcription regulatory region sequence-specific binding, and other processes (Fig. 5C).

Figure 5. GO enrichment analysis of differentially expressed genes. (A) Bubble plots of the enrichment analysis of differentially expressed genes in biological processes between the control and model group. (B) Bubble plots of the enrichment analysis of differentially expressed genes in cell composition between the control and model group. (C) Bubble plots of the enrichment analysis of differentially expressed genes in molecular function between the control and model group. (D) Bubble plots of the enrichment analysis of differentially expressed genes in biological processes between the model and FN group. (E) Bubble plots of the enrichment analysis of differentially expressed genes in cell composition between the model and FN group. (F) Bubble plots of the enrichment analysis of differentially expressed genes in molecular function between the model and FN group. N = 3. GO, Gene Ontology; FN, fibrauretine.

The differentially expressed genes were highly enriched in the development of visual perception and response to amphetamine among the biological processes, when comparing the model group with FN group (Fig. 5D). Among the cellular components, differentially expressed genes were significantly enriched in extracellular space, extracellular region, glutamatergic synapse and photoreceptor outer segment (Fig. 5E). Among the molecular functions, they were significantly enriched in calcium ion binding, sequence-specific DNA-binding, RNA polymerase II transcription regulatory region sequence-specific binding, etc. (Fig. 5F).

KEGG enrichment analysis of differentially expressed genes: For the purpose of better comprehending the biological functions performed by differentially expressed genes, we annotated the differentially expressed genes of the control, FN and model groups with the KEGG database for analysis, mainly comparing to environmental information processing, cellular processes, human diseases and organic systems, genetic information processing, among which the genes enriched to human diseases were the most abundant, 7 items in the FN and model groups are associated with AD. In environmental information processing, the model/control and FN/model comparison groups enriched 17 and 15 pieces of information related to the PI3K-AKT signaling pathway. The KEGG classification annotation map is shown in Fig. 6A, B.

Figure 6. KEGG enrichment analysis. (A) KEGG classification annotation map of differentially expressed genes between the control group and model group. (B) KEGG classification annotation map of differentially expressed genes between the model group and FN group. (C) Top 20 pathways enriched to KEGG in the control group vs. model group. (D) Top 20 pathways enriched to KEGG in the model group vs. FN group. N = 3. KEGG, Kyoto Encyclopedia of Genes and Genomes; FN, fibrauretine.

Using KEGG classification annotation analysis, we selected the top 20 significant signaling pathways for KEGG enrichment analysis. The findings showed that the differentially expressed genes in PI3K-AKT signaling pathway, human papilloma virus infection, MAPK signaling pathway and cAMP signaling pathway were significant, as shown in Fig. 6C, D. We noticed that the differentially expressed genes were more significantly expressed in the PI3K-AKT signaling pathway and MAPK signaling pathway after FN treatment. As a result, it is inferred that FN may exert its therapeutic effect on AD via the PI3K-AKT signaling pathway or MAPK signaling pathway.

Gene expression validation results

In order to verify the feasibility of transcriptome results, we randomly selected SPP1, KLF4 and Nr4a1 genes related to the disease-related for RT-qPCR verification. We found that the expression of KLF4 and Nr4a1 was significantly decreased in the model group (p < 0.01), and a significant increase in the expression of these genes after FN treatment (p < 0.05). In addition, the model group exhibited high expression of SPP1, which was drastically decreased following FN treatment (p < 0.01, Fig. 7). The RT-qPCR findings are in line with the outcomes of the transcriptome, proving that the results of transcriptome sequencing are feasible.

Figure 7. The mRNA expression level of SPP1, Nr4a1, KLF4 in each group was detected by RT-qPCR. Values are presented as mean ± SD. N = 6. FN, fibrauretine. ##p < 0.01 vs. control group. **p < 0.01, *p < 0.05 vs. model group.

Differentially expressed proteins analysis

Protein expression analysis: Principal component analysis was performed on each group in order to gain a preliminary understanding of the overall differences between groups and the magnitude of variability across samples within groups. Fig. 8A presents the findings. The scatter points corresponding to the samples of several groups showed mutual aggregation in the group, indicating that the repeatability in the group was better, the sample data was very similar, and there was a good degree of discrimination between the groups, indicating that there was a huge difference between the samples of the control group, the model group and the FN group.

Figure 8. PCA analysis and differentially expressed proteins volcano map, Wayne map and heat map. (A) PCA analysis between groups. (B) Differentially expressed proteins volcano map between the control group and model group. (C) Venn of common differentially expressed proteins in the control group and model group, model group and FN group. (D) Differentially expressed proteins volcanoes between the model group and FN group. (E) Differentially expressed proteins clustering heat map. N = 3. PCA, principal component analysis; FN, fibrauretine.

Results of protein difference analysis: In the control group in contrast to the model group, there were 300 different proteins, of which 157 proteins were down-regulated and 143 proteins were up-regulated (Fig. 8B). 565 differentially expressed proteins were found when comparing the model group to the FN group, of which overall 289 proteins were expressed up-regulated and 276 proteins were expressed down-regulated (Fig. 8C). We could see that there were significant changes in differentially expressed proteins following FN treatment. Among the differentially expressed proteins in two comparative groups, we identified a common differentially expressed proteins (Fig. 8D), among the 129 differentially expressed proteins, the expression trend of 117 differentially expressed proteins was opposite, and the expression trend of 12 differentially expressed proteins was the same, and the specific information is listed in Supplementary Table 2. Protein with the same or similar expression patterns in different samples were clustered and displayed through heat maps (Fig. 8E).

Differentially expressed proteins GO enrichment analysis: The results showed that, in contrast to the model and control groups, the differentially expressed proteins engaged in molecular functions were primarily involved in mRNA splicing, via spliceosome (Fig. 9A). Significantly enriched in biological processes including DNA binding (Fig. 9B), and involved in cellular composition including glutamatergic synapse, nuclear speck, catalytic step 2 spliceosome, and so on (Fig. 9C).

Figure 9. GO enrichment analysis of differentially expressed proteins. (A) Control group vs. model group in terms of molecular function enriched to entries. (B) Control group vs. model group enriched to entries on biological processes. (C) Entries enriched to cell composition in the control group vs. model group. (D) The entries enriched in molecular function in the model group vs. FN group. (E) Entries enriched to biological processes in the model group vs. FN group. (F) Entries enriched to cell composition in the model group vs. FN group. N = 3. GO, Gene Ontology; FN, fibrauretine.

In contrast to the model group, the FN group’s differentially expressed proteins were primarily involved in molecular functions such as RNA binding, calcium-transporting ATPase activity (Fig. 9D), and the differentially expressed proteins engaged in biological processes mainly included in mRNA splicing, via spliceosome, mRNA processing, etc. (Fig. 9E). In cell composition, ribonucleoprotein complex and nuclear speck were highly enriched in the differentially expressed proteins (Fig. 9F). These findings offer a critical foundation for further investigation into FN’s mechanism of action in AD.

Functional annotation and enrichment analysis of the differential protein KEGG: The annotation of the function of the protein itself alone does not provide a better representation of the function of the differentially expressed proteins, so we utilized the KEGG databaseenriched to enrich differentially expressed proteins in cellular processes, environmental information processing, genetic information processing, human diseases, examine metabolism and organismal systems. The findings demonstrated that metabolism pathways were more considerably enriched in the model group in contrast to the control group. Meanwhile, there are 9 pieces of information related to AD in human diseases, as well as PI3K-AKT, cAMP, and calcium signaling pathways, etc. (Fig. 10A). The differentially expressed proteins were also significantly enriched in the metabolic pathway, PI3K-AKT signaling pathway, pathways in cancer, calcium signaling pathway, and cAMP signaling pathway in the FN group in comparison to the model group (Fig. 10B). It can be seen that the differentially expressed proteins between the contorl group and model group are significantly enriched in AD, which is as expected, proving the feasibility of our experiment. The differentially expressed proteins between the model/control groups and FN/model groups were significantly enriched in the PI3K-AKT signaling pathway, which provides us with a basis to validate the mechanism of AD treatment with FN.

Figure 10. KEGG classification of differentially expressed proteins in each group. (A) KEGG classification of differentially expressed proteins between the control group and model group. (B) KEGG classification of differentially expressed proteins between the model group and FN group. N = 3. KEGG, Kyoto Encyclopedia of Genes and Genomes; FN, fibrauretine.

Western blot detection of PI3K-AKT pathway-related protein expression

The results of proteomic and transcriptomic sequencing analysis showed that the PI3K-AKT pathway was frequently present, which tends to indicate that the mechanism of AD treatment by FN is most likely via the PI3K-AKT signaling pathway. Western blot experiments were performed and the findings are depicted in Fig. 11. PI3K-AKT pathway-related protein content was much higher in the model group than the control group, and this trend was significantly down-regulated by FN treatment, which is in line with our transcriptomic and proteomic sequencing results.

Figure 11. The effect of FN on the expression of PI3K-AKT pathway-related proteins. (A) Western blot was used to detect the expression of PI3K-AKT pathway-related proteins. (B) The level of PI3K. (C) The level of AKT. (D) The level of MTOR. Values are presented as mean ± SD. N = 3. FN, fibrauretine. ##p < 0.01, #p < 0.05 vs. control group. *p < 0.05 vs. model group.

Combined analysis of differentially expressed genes and differentially expressed proteins

In order to further understand the mechanism of FN in the treatment of AD, we performed overlapping analysis of the enriched differentially expressed genes and differentially expressed protein-coding genes, and found a total of 19 overlapping genes, including 14 genes with the same expression trend. The up-regulated genes were Prex2, Grm2, Dpysl4, Chl1, Alb, and the down-regulated genes were Vat1, Th, Sqor, SPP1, Scg2, RGD1311744, Enpp6, Baiap3, Ass1. There are 5 genes with different expression trends, namely Rpl17, Hbb, and Hba-a3. Cbln1, Calb2. The results are shown in Fig. 12 and Table 5.

Table 5 . The expression trend of overlapping genes.

Differentially expressed
protein-coding genes
FDR_0.05_FC_1.5_regulatedDifferentially expressed genesFDR_0.05_FC_1.5_regulated
Vat1DOWNVat1DOWN
ThDOWNThDOWN
SqorDOWNSqorDOWN
SPP1DOWNSPP1DOWN
Scg2DOWNScg2DOWN
RGD1311744DOWNRGD1311744DOWN
Enpp6DOWNEnpp6DOWN
Baiap3DOWNBaiap3DOWN
Ass1DOWNAss1DOWN
Cbln1UPCbln1DOWN
Calb2UPCalb2DOWN
Rpl17DOWNRpl17UP
HbbDOWNHbbUP
Hba-a3DOWNHba-a3UP
Prex2UPPrex2UP
Grm2UPGrm2UP
Dpysl4UPDpysl4UP
Chl1UPChl1UP
AlbUPAlbUP

FC ≥ 1.5 and FDR < 0.05 set as the threshold for significant differential expression. N = 3. FC, fold change; FDR, false discovery rate.



Figure 12. Overlapping of differentially expressed protein-coding genes and differentially expressed genes. N = 3.

Modern studies have shown that there is a link between Th, Scg2, Rpl17, Chl1, Alb and AD [15-19], SPP1, Th, Prex2 play a role in a variety of diseases by regulating the PI3K-AKT signaling pathway [20-23]. In the transcriptome sequencing, the KEGG enrichment analysis of differentially expressed genes was significantly enriched in the PI3K-AKT signaling pathway. In the proteomics sequencing, the KEGG enrichment analysis of differentially expressed proteins was also significantly enriched in the PI3K-AKT signaling pathway. The results of overlapping analysis of transcriptomics and proteomics are the same as those obtained in our transcriptomics and proteomics analysis. The PI3K-AKT signaling pathway seems to be inseparable from the treatment of AD with FN. The overlap between differential proteins and differential genes was screened by joint analysis, which provided a basis for further exploration of the mechanism of FN in AD.

AD is a neurodegenerative disease caused by multiple complex factors whose pathogenesis is unclear. Further research and discussion are still needed [24]. Currently, plenty of drugs exist in the market for the treatment of AD with a single type, which can only alleviate the symptoms to a certain extent but possess side effects [25]. FN has recently been extensively utilized, extensive studies on it have found that FN has good pharmacological effects such as anti-inflammatory, antioxidant, anti-viral, anti-aging, and neuroprotective effects [26]. This points out that FN has the potential in improving cognitive dysfunction in AD [27]. In this study, an AD model was constructed by intracranial injection of Aβ1-40, treated by gavage with FN. Morris water maze experiment showed that the AD model was successfully established, and FN treatment could restore the learning and memory ability of rats. The ELISA kit was employed for detecting the FN increased ACh content in brain tissue, decreased AChE activity, and improved cholinergic dysfunction in AD rats. Previous studies discovered that Aβ deposition and hyperphosphorylation of tau protein in the brain equally aggravated the onset as well as the development of AD disease. In our study, the immunohistochemical results showed that FN lowered the expression of P-Tau and Aβ protein in AD rat’s hippocampus and maintained normal neurological function. Our study confirms FN’s role in the cure and prevention of AD, providing a strong basis for the next in-depth study.

In recent years, histology has been widely used as an entry point to study the treatment of diseases by drugs [28]. In transcriptomic studies, when RNA is abnormally expressed, it leads to a large accumulation of P-Tau and Aβ. This further promotes the development of AD, suggesting that key transcriptomic factors may offer new ideas along with the targets for AD therapy. Therefore, we selected FN, control, and model groups of AD model rats for transcriptomic analysis [29]. KEEG analysis revealed that the differentially expressed genes in the model group compared with the control group and the FN group compared with the model group were co-enriched in PI3K-AKT signaling pathway, MAPK signaling pathway. In addition, among the differentially expressed genes between the control group and model group, as well as between the model group and the FN group, there are a total of 199 identical differentially expressed genes. We randomly screened three differentially significant genes for RT-qPCR validation, SPP1 is secreted phosphoprotein 1, which is released by perivascular macrophages and perivascular fibroblasts in the AD mouse model. Subsequently, SPP1 induces microglia in the brain parenchyma to phagocytose neuronal synapses [30]. A study has found that SPP1 leads to the upregulation of PD-L1 expression, mediates the polarization of macrophages, reduces SPP1 gene expression, and can promote apoptosis by inhibiting PI3K-AKT signaling pathway [31]. RT-qPCR findings highlighted that SPP1 expression was considerably decreased in the FN group in contrast to the model group, suggesting that FN may promote necrotic cells or lesions by inhibiting the expression of SPP1 cell apoptosis to play a role in the treatment of AD. Nr4a1 is a member of the Nr4a subfamily of isolated nuclear receptors in the steroidal thyroid receptor family. Studies have claimed that Nr4a1 has become a key transcriptional regulator in the role of cytokines and growth factors affecting our aging population [32,33]. It has been reported that the NR4A nuclear receptor subfamily is a potential therapeutic target for the treatment of age-related diseases such as diabetes and neurodegenerative diseases. The NR4A receptor is part of the DNA repair mechanism and promotes DNA repair. Members of the NR4A subfamily should also be involved in the anti-aging properties of stimulants, as these receptors are induced by various forms of cellular stress and stimulate protective cellular responses such as antioxidant activity and DNA repair [34]. RT-qPCR findings indicated that Nr4a1 expression was much greater in the FN group in contrast to the model group, pointing out that FN might have a part in the treatment of AD by activating Nr4a1 expression to promote apoptosis. KLF4 is Krüppel-like factor 4, it well established in regulating cell differentiation and reprogramming, was selectively diminished in aged macrophages [35]. KLF4 is a tumor suppressor gene involved in regulating cellular senescence, promoting apoptosis, regulating tumor cell invasion and metastasis, as well as inhibiting tumor cell growth. It has a number of inhibitory effects on tumorigenesis and progression. KLF4 can inhibit the expression of the PI3K-AKT signaling pathway while further promoting autophagy [36]. The RT-qPCR findings indicated that KLF4 expression was much greater in the FN group in contrast to the model group, emphasizing that FN might have a part in AD treatment by promoting autophagy through KLF4. The outcomes of RT-qPCR validation of the screened differentially expressed genes were in line with the transcriptome sequencing results, demonstrating the feasibility of the transcriptomic outcomes.

In recent years, not only transcriptomics has been well studied, but also proteomics is emerging as one of the effective methods to discover disease biomarkers and predict disease-related targets for treatment. This provides evidence for studying the pathogenesis of AD at a deeper level. For instance, Andreev et al. [37] carried out a quantitative proteomic analysis of the cerebral cortex of AD patients and normal patients, which showed that the levels of up to nearly 200 of these proteins differed significantly between AD patients and normal patients [37,38]. There is now a growing body of data validating the elevated expression of differentially expressed proteins in the AD brain [39].

In this study, GO enrichment analysis indicated that differentially expressed proteins in three groups were primarily enriched in calcium-transporting ATP activity and RNA binding regarding molecular functions. KEGG enrichment analysis highlighted that the differentially expressed proteins in the model group compared with the control group and the FN group compared with the model group were mostly enriched in the metabolic pathway, AD, PI3K-AKT signaling pathway. PI3K/Akt pathway is an important signal transduction pathway in the body. It plays an important role in cell survival, proliferation, apoptosis and other biological activities [40]. It is closely related to neuronal apoptosis and oxidative stress in brain tissue of AD patients. PI3K generates phosphatidylinositol diphosphate by phosphorylating the 3-hydroxyl group on the inositol ring, and then transmits the signal to the downstream protein and activates Akt. The activated PI3K/Akt pathway has the effects of promoting cell survival and anti-apoptosis [41]. Western blot experiment was used to test the expression of PI3K-AKT signaling-related proteins. The results revealed that both PI3K, AKT and MTOR protein expression was significantly reduced following FN treatment in contrast to the model group, suggesting that FN may improve AD symptoms by inhibiting PI3K-AKT signaling, which is overexpressed in AD, and reducing the expression level of MTOR, thereby promoting the clearance of pathological proteins by cellular autophagy. In addition, we carried out overlapping analysis of the enriched differentially expressed protein-coding genes and differentially expressed genes, and found a total of 19 overlapping genes, most of them are related to AD and PI3K-AKT signaling pathways. We conclude that the FN mechanism of action for AD may be through the regulation of SPP1, Nr4a1, Prex2, KLF4 and other genes expression so as to further inhibit PI3K-AKT signaling pathway expression, to exert neuroprotective effects.

In conclusion, we potentially evaluated the therapeutic effects of FN in AD model rats, effectively alleviated abnormalities in choline function, reduced Aβ and P-Tau protein expression, and treated learning and memory abilities and cognitive dysfunction in AD rats. Proteomic and transcriptomic analysis, in combination with Western blot and RT-qPCR, showed that FN maybe regulating the expression of SPP1, Nr4a1, Prex2 and KLF4 in order to further inhibit the PI3K-AKT signaling pathway, to exert neuroprotective effects, and to reveal FN as potential candidates for AD treatment, to offer a theoretical foundation for subsequent studies.

This work was supported by the [Jilin Science &Technology Development Plan] under Grant [number: 20220304002YY], we thank Biomarker Technologies for assisting in sequencing and bioinformatics analysis.

This work was supported by the [Jilin Science &Technology Development Plan] under Grant [number: 20220304002YY], demonstration and promotion of sika deer breeding and reproduction, disease prevention and control, product processing industry technology [number: 202300801-04], the Jilin Province Aid Project for Xinjiang Uygur Autonomous Region [number: 0207-202020043].

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