Positive Effects of Walnut Meal Extracts on Memory Impairment of Alzheimer's Disease Mice Induced by D-Galactose

Walnut kernel was a traditional Chinese medicine, as well as a brain power boosting food. Walnut meal (WM) was prepared from walnut kernel by cold-press deoil, which was rich in polyphenols. In this study, we investigated the positive effects of walnut meal extracts on memory impairment of Alzheimer's disease (AD) mice induced by D-galactose. After 6 weeks of WMP treatment, the behavioral results showed that WMP could signicantly improve the learning and memory of D-galactose Model (MOL) mice. The biochemical assay showed WMP could increase the amount of Acetylcholine (Ach) and reduce the oxidative stress and inammation. Meanwhile, WMP improved the expression of neural stem cells, restored the number and the shape of neurons. The RNA-seq analysis revealed 284 differentially expressed genes in the hippocampus were regulated by WMP treatment, among which Gzma, Apol11b, H2-Q6 were up-regulated, while Rny1, Scgn, Col6a3 were down-regulated. Further analysis disclosed the differentially expressed genes were relevant to PI3K-Akt signaling pathway, FoxO signaling pathway, PPAR signaling pathway, neuroactive ligand-receptor interaction, cellular senescence, and particularly strongly interacted with the ribosomal family genes (Rpl35a, Rps27rt, Rpl3l, Rpl21, Rpl26). In addition, transcription factor Ep300 regulated genes of Cdkn1a, Spp1, and Tnfsf10 distinctly in the hippocampus, which were involved in inammation and protein kinase. Transcription factor Pparg regulated genes of Angptl4, Fabp4, and Plin4, which were mainly expressed in PPAR (Peroxisome Proliferator-activated receptors) signaling pathway. This study might assist in identifying new targets to restore memory impairment in Alzheimer's disease. This study has explored the therapeutic effect of WMP on memory impairment in AD mice and analyzed changes of gene expression in the hippocampus. Behavioral experiment illustreated that WMP could signicantly improve the memory impairment of MOL mice, boosting spatial learning, memory, and recognition abilities. Hernandez-Rabaza et al. found apoptosis of hippocampus neurons was an important reason for the decline of learning and memory ability 20 . Slice analyses of hippocampus stained with Nestin (a marker of neural stem cells) showed the number of neurons were signicantly decreased, which could be improved by WMP. Neural stem cells regulated nervous system homeostasis through self-renewal and multi-directional differentiation 21 . The hippocampus played an essential role in the process of learning and memory 22 . Since the neural stem cells in which could produce new neurons and glial cells to maintain the function of the hippocampus 23 . Previous studies had reported inammation and oxidative stress were crucial risk factors for Alzheimer's disease, and the inammatory factor occurred in pathologically vulnerable regions of AD 24 . Our results revealed an imbalanced brain redox in MOL mice, as well as the decreased oxidoreductase forming lipid peroxide (MDA) and activated inammation factor (IL-6). Hence, WMP displayed a powerful ability to reduce oxidative stress and anti-inammation.


Introduction
Alzheimer's disease (AD) was the most common neurodegenerative disorder with memory, judgment, and abstract thinking disorders in the clinic 1 . With the increasing aging population around the world, AD has become a world-recognized medical problem with an incidence of more than 50 million patients 2 . The pathological features of AD usually include amyloid beta protein (Aβ) plaque deposition, hyperphosphorylation of tau protein, activation of microglia, and neurons apoptosis 3 . Recent studies have revealed the correlation between AD and the in ammation process as well as oxidative stress.
Oxidative stress was the main cause of aging due to the imbalance between the production and scavenging of active nitrogen free radicals (RNS) and reactive oxygen radicals (ROS). So the reduction of cellular expression, the restrained activity of antioxidant proteins, and consequently the augment of oxidative stress would play a central role in AD. The animal model of AD illustrated that immune cells in the brain could affect neurodegenerative diseases 4 . At present, two types of chemical synthetic drugs, Cholinesterase inhibitors (ChEIS) and Memantine, have been used to treat AD. ChEIS therapeutics was based on the cholinergic hypothesis, that AD patients were usually accompanied by cholinergic system dysfunction to some degree while ChEIS was able to promote the neurotransmission of cholinergic by inhibiting the activity of ACHE. The other chemotherapeutic, memantine, could bind to an ionotropic glutamate receptor NMDAR non-competitively. The activation of NMDAR would unselectively open a cation channel with high Ca 2+ permeability, causing continuous activation of NMDA receptors 5 .
Rregrettably, the AD medicines were not only with less choices but also with unapparent effect. Dgalactose injection in mice could induce memory impairment accompanied by other pathological features of AD, such as brain oxidative damage, neuroin ammation, cholinergic damage 6 , and decreasing of cortical neurons and hippocampal pyramidal cells 7 ; 8 .
Walnut, a major nut worldwide with high nutritional value, of which the kernel was rich in oils, functional fatty acids, and phenolic compounds such as avonoids, phenolic acids, and tannins 9 . After exctracting oil from the walnut kernel by cold pressing, walnut meal (WM) has been left as residue which has not been fully utilized. The walnut meal extracts (WMP) were rich in polyphenols, which have shown the signi cant activites of anti-oxidation, anti-aging, hypolipidemic, and improvement of learning and memory 10 . The polyphenol substances of WMP identi ed by LC/MS were including gallic acid, ellagic acid, chlorogenic acid, glansreginin A, glansreginin B, rutin, vanillic acid glucoside, cumaroylquinic acid, and digalloyl glucose 11 . The purpose of this study was to explore the therapeutic effects of WMP on memory impairment of AD mice induced by D-galactose. In addition, the RNA transcriptome was used to analyze the differentially expressed genes in the hippocampus of AD mice treated by WMP.

Preparation of extracts from Walnut meal (WMP)
The walnut meals (WM) were extracted twice for 60 minutes with 50% ethanol (1:50, W/V) under re ux, and the ltrate was concentrated by Rotary Evaporator to obtain the extracts (WMP).
After a week of adaptive feeding, the experimental mice were randomly divided into 4 groups: control group (CD, n=20), D-galactose Model group (MOL, n=20), D-galactose + walnut meal extracts group (WMP-L, 800 mg/kg/d, n=20) and D-galactose + walnut meal extracts group (WMP-H, 1600 mg/kg/d, n=20). Except for the CD group, all the other groups were subcutaneously injected with D-galactose (200 mg/kg/d) for 12 weeks, and the D-galactose was dissolved in saline, while the CD group was subcutaneously injected with 0.9% normal saline (0.1 ml/10 g) every day. The WMP-L and WMP-H groups were given walnut meal extracts by intragastric administration since the eighth week, and the CD and MOL groups were intragastric administered of the same volume of normal saline. The mice were carried out in a 25 × 25 ×30cm black square box. First, the mice were put into the new object recognition device and allowed to explore freely for 5 minutes. Second, the mice were placed to the new object recognition device containing two identical objects (A + B) and allowed mice to explore freely for 5 minutes on the next day. Last, object B was replaced with a new object C in a new object recognition device, and the mice were placed to a new object recognition device to explore for 5 minutes, the exploration time of different objects (A + C) was recorded on the third day. Recognition index was used to evaluate the new object recognition ability, which was de ned as the difference between exploration time of novel and familiar object, and dividing the total time spend exploring two objects in the test phase 12 .

Morris Water Maze Test
Morris Water Maze test used a black round pool with water (20 ± 1 °C), a depth of 45 cm, surrounded by water maze clues, and a hidden platform at 1.0 cm underwater. The MWM was divided into four quadrants, and the hidden platform was located in the fourth quadrant. The mice were placed into the water facing the wall from one of the four quadrants (Once in each quadrant) for 4 days. If the mouse successfully reached the platform within 60 s, which was allowed to stay on the platform for 15 s, if the mouse could not nd the platform within 60 s, the mouse was guided and stayed on the platform for 15 s. During the testing period, the hidden platform was removed and the mouse was placed in the pool on the wall in the quadrant opposite the platform, the track and time in each mouse in each quadrant was recorded during the 60s 13 .

Determination of organ index
The mice were fasted overnight and euthanized with pentobarbital sodium (40 mg/kg) at the end of the experiment. The spleen, brain, liver, and heart were separated and washed with frozen saline and weighed these organs to calculate the organ coe cient. The organ index was de ned as coe cient (mg/g) = organ weight (mg) / body weight (g) 14 .
Biochemical assay The brain tissue of mice was homogenized in order to determine the biochemical indexes. The mouse brain was homogenized, and then the homogenate centrifuged for 15 min (3500 g). The supernatant was collected to determine the biochemical indexes. The levels of SOD, MDA, Ach, AchE, and IL-6 in the brain were detected by the kit instructions.

Nissl staining
The brains of the mice were completely stripped off, then xed with 4% paraformaldehyde, embedding in para n, cutting into 30 μm thick brain slices. The brains were washed twice with 1 × PBS solution with the sections stained by 0.5mL Nissl solution and incubated for 15 min at 37 ℃. Fixed the slides into 95% alcohol and dehydrated twice for 2 min each time, then put the dehydrated slides into xylene for 2 min each time 15 . The number, morphological structure, and color of Nissl bodies in the area were observed by taking pictures of 40 times the size under a microscope.

Immunohistochemistry Analysis
The brains were completely stripped out from mice, and xed in 10% paraformaldehyde after which they were dehydrated in ethanol and embedded in para n. The brain slices (30μm) were cut by tissue slicer, which were stained with Nestin by immunohistochemistry, dewaxing in xylene and different concentrations of ethanol, repairing solution by repairing antigen, PBS cleaning 3 times, adding Nestin antibody and incubate overnight at 4 ℃, PBS cleaning 3 times. The brain slices were stained with DVB for 15min, distilled water was added to stop, the brain slices were added to hematoxylin liquid for staining. In each slice, three visual elds in the hippocampus were photographed at 400 × magni cation. The brain slices were determined using Image-Pro Plus 6.0 and the average integral optical density (IOD) was used to evaluate the expression of Nestin in neural stem cells 16 .

RNA extraction, library preparation, and sequencing
In the experiment, total RNA in the hippocampus of CD, MOL, and WMP groups were extracted with triazole reagent, the integrity and concentration of total RNA were tested by Agilent Technologies 2100 RNA Nano 6000 Assay Kit 17 . According to the manufacturer's recommendations sequencing libraries were generated using NEB Next® Ultra TM RNA Library Prep Kit for Illumina® (NEB, USA), mRNA was randomly interrupted into fragments of 200bp. The strand cDNA was synthesized by random hexamer primers and M-MuLV reverse transcriptase using mRNA as template, and second-strand cDNA synthesis, cDNA adds End Repair Mix to make its at end, then add A tail to connect the sequencing connector at 3 ' . The cDNA was puri ed using the AMPure XP system (Beckman Coulter, Beverly, USA) to select cDNA fragments of 200~300 bp in length, the cDNA was enriched by using PCR ampli cation. Finally, the library was sequenced with high throughput using Illumina HiSeq4000, and the sequencing length is 150bp 18 .

Quality control of reads
Quality control of Raw Data in FASTQ format in order to obtain Clean Data that can be used for subsequent analysis, the Clean Reads was sequenced with the designated mouse genome using software for sequence alignment of the second generation (HISAT2) to obtain its location information on the reference genome 19 .

Differential gene expression analysis
The feature Counts software was used to calculate the FPKM (expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced) of genes expression in the hippocampus of all groups, the DESeq2 software was used to analyze the differential expression of gene (DEGs) by reading count data, genes with a |log2 (fold change)| ≥ 1 and p-value < 0.05 were de ned as difference signi cance.
Bioinformatics analysis of the differential expressed genes For visualization, the volcano map and heat map were constructed using the dnet R package. The Gene Ontology, (GO, http://www.geneontology.org/) was used to analyze the biological function of DGEs, and Kyoto Encyclopedia of Genes and Genomes (KEGG) was selected to analyze the pathway of DEGs. The STRING database was used to build a PPI network, which visual analysis was carried out by Cytoscape software.

Statistical Analysis
The GraphPad Prism 7.0 software was used for statistical analysis. Behavioral tests data were expressed as the mean ± SEM, other data expressed as the mean ± SD. The data were analyzed by One-Way ANOVA following by Duncan's post-hoc test at the signi cant level of 0.05.

Results
Effect of WMP on behavioral experiment of Alzheimer's disease mice induced by D-galactose Morris water maze is mainly re ecting the spatial learning and memory ability of animals. Fig.1A illustrated the escape latency of three groups continuously shortened with the increase of training days.
The MOL mice exhibited longer latency compared with CD mice from the second day, and WMP signi cantly restored the escape latency in MOL mice. On the fth day, the platform was removed to conduct a positioning cruise test. The MOL mice took much longer time to nd the hidden platform and stay less time in the target quadrant ( Figure.1B-C). Both parameters could be signi cantly improved after WMP treatment in MOL mice. The swimming records illustrated that the swimming trajectory of MOL mice in the target quadrant was less than CD mice, however, which was signi cantly recovered after WMP treatment ( Figure.1D). In the new object experiment, the recognition index of MOL mice was signi cantly lower than CD mice, while treatment with WMP restored the recognition index and improved their novelty cognitive ability in MOL mice ( Figure.1E).

Effect of WMP on Organ Index of Alzheimer's disease mice induced by D-galactose
At the end of the experiment, the brain index, heart index, spleen index, liver index of MOL group have signi cantly decreased compared with CD group. Compared with MOL group, treatment with WMP could increase the brain index, spleen index, and liver index (Table 1). Effects of WMP on Ach, AchE, and IL-6 of the brain in Alzheimer's disease mice induced by D-galactose The AchE and IL-6 levels in the brain of MOL mice were signi cantly increased, while the Ach level was lower (p < 0.05), compared with CD group ( Figure. 2A-C). In contrast, treatment with WMP would decrease the AchE and IL-6 levels, and increase Ach level in the brain of MOL mice (p < 0.05 for all).

Effects of WMP on brain oxidative damage in Alzheimer's disease mice induced by D-galactose
Oxidative damage was closely related to Alzheimer's disease. SOD level in MOL group was signi cantly lower than CD group (p < 0.05) (Figure. 2D). In contrast, only WMP-L administration would increase SOD level in MOL mice (p < 0.05). MDA level signi cantly increased in MOL group vs. CD group (p < 0.05). However, treatment with WMP could decrease MDA level in the brain of MOL mice (p < 0.05) ( Figure. 2E).
Effect of WMP on brain histopathology of Alzheimer's disease mice induced by D-galactose As seen in Figure.3, histopathological observation showed that the Nissl corpuscles of the neurons in hippocampus DG were decreased, the color of plasma became darker, and the structure of Nissl corpuscles was abnormal in MOL mice, it's the hippocampus CA3 that the Nissl corpuscles of the neurons were partially dissolved or disappeared, and the number of neurons signi cantly decreased. However, WMP treatment could increase the number of Nissl bodies in hippocampus (CA3, DG) neurons, the hippocampus (CA3, DG) neurons were orderly arranged with a signi cant increase neuron cell number.

Effect of WMP on Hippocampus Neural Stem cells of Alzheimer's disease mice induced by D-galactose
It has been found that neurodegenerative disease is caused by neuronal degeneration, senescence, loss, and death of neurons. NScS (senescence of neural stem cells) is an important factor of neurodegenerative disease, while Nestin is a classic NSCS marker. Figure.4 illustrated that the expression of Nestin in the hippocampus (CA3, DG) of MOL mice signi cantly decreased (p < 0.001). However, the expression of Nestin in hippocampus (CA3, DG) neurons of MOL mice were increased after WMP treatment, indicating that WMP treatment could promote the proliferation of hippocampus neural stem cell.

Effect of WMP on Hippocampus Neural Stem cells of Alzheimer's disease mice induced by D-galactose
It has been found that neurodegenerative disease is caused by neuronal degeneration, senescence, loss, and death of neurons. NScS (senescence of neural stem cells) is an important factor of neurodegenerative disease, while Nestin is a classic NSCS marker. Figure.4 illustrated that the expression of Nestin in the hippocampus (CA3, DG) of MOL mice signi cantly decreased (p < 0.001). However, the expression of Nestin in hippocampus (CA3, DG) neurons of MOL mice were increased after WMP treatment, indicating that WMP treatment could promote the proliferation of hippocampus neural stem cell.

Effect of WMP on DEGs in the hippocampus of Alzheimer's disease mice induced by D-galactose
The cDNA libraries from CD, MOL, and WMP (n = 3) hippocampus of the brain were prepared and sequenced. The good quality of RNA with favorable purity was used to build the sequencing library. The quality control results showed that RNA bands were clear and no other impurities were contaminated, OD260/280 ≥ 1.9, OD260/230 ≥ 1.9, RIN ≥ 8.6. Through RNA sequencing, we achieved RNA sequencing throughput of more than 5.71GB in each hippocampus sample, and 90% of Unique reads aligned to the reference genome (Table 2).
Furthermore, to eliminate individual-speci c transformation, the hippocampus from three mice in the same group were used. Venn diagram analysis showed the uniquely expressed genes and commonly expressed in CD, MOL and, WMP groups, the number of commonly expressed genes among the CD, MOL, and WMP groups were 19888, of which 594 genes were expressed in the CD and MOL groups and 115 genes were expressed in the WMP and MOL groups ( Figure 5A). The differential expressed genes (DEGs) were de ned as those with |log2(fold change)| ≥1 and p < 0.05, the DEGs were further analyzed by constructing volcanic maps. The red dots correspond to signi cantly up-regulated transcripts (p < 0.05); the green dots correspond to signi cantly down-regulated transcripts (p < 0.05); and the gray dots correspond to no stat. Figure 5B instructed that 633 genes were differentially expressed in the hippocampus of MOL group relative to CD group, where 264 were up-regulated and 369 were downregulated. Compared with MOL group, treatment with WMP could regulate 284 differentially expressed genes, where 126 were up-regulated and 158 down-regulated ( Figure 5C). In addition, cluster analysis using Euclidean distance and a hierarchical algorithm was used to show the differentially expressed genes of CD, MOL, and WMP groups. The clustering analysis showed that there was a similarity of gene expression between CD, and WMP groups. However, the gene expression of MOL group was different from CD and WMP groups ( Figure. 5D). What's interesting was that compared with the CD group, 15 genes in the hippocampus of MOL mice were up-regulated, however, which were down-regulation after the WMP treatment. In addition, 20 genes of the hippocampus in MOL mice were down-regulated compared with the CD group, which were up-regulated after the WMP treatment. It is suggested that WMP treat memory impairment mainly by altering the hippocampus with these genes, which include H2-Q6

Bioinformatics analysis of DEGs in the hippocampus of AD mice induced by D-galactose
To investigate the GO functional of the DEGs, we performed a functional classi cation according to Gene Ontology terms ( Figure. 6A), the primary biological processes in the hippocampus affected by WMP included response to light stimulus, positive regulation of response to cytokine stimulus, hypoxanthine oxidation, positive regulation of signal transduction and in ammatory response. The most affected cellular component in the hippocampus were extracellular space, extracellular region. The molecular function associated in the hippocampus with WMP treatment were TAP binding, antigen binding, amide binding. KEGG analysis provided a platform for the systematic analysis of gene function in the networks of gene products. The main signi cant pathways in the hippocampus affected by WMP were related to immune, in ammatory and nerve, including ribosome, antigen processing and presentation, PPAR signaling pathway, tyrosine metabolism, cellular senescence, FoxO signaling pathway, linoleic acid metabolism, and so on ( Figure. 6B). To further analyze the relationships among the DEGs [|log2(fold change)| ≥ 1 and p < 0.05] in the hippocampus affected by treatment with WMP, PPI was performed using STRING database and interpreted with Cytoscape 3.6.0 ( Figure. 7A). The blue circles correspond to signi cantly up-regulated genes by treatment with WMP, pink circles correspond to signi cantly downregulated genes by WMP, and the size of the circle represents the multiple to be adjusted. In the hippocampus, 86 differentially expressed genes were connected with each other, the top 10 genes that were highly connected to other genes in PPI including Srp54a, Rpl3l, Rpl21, Rpl26, Gm10320, Gm7536M, Rpl30, Rpl35a, Rps27rt, Sec61g, which mainly existed in the ribosomal family. Ribosomal family genes (Rpl35a, Rps27rt, Rpl3l, Rpl21, Rpl26) were remarkably adjusted after administration of WMP (Fig. 7B). Further analyses of the transcription factors of these genes showed Cdkn1a, Spp1, Tnfsf10 were obviously regulated by upstream transcription factor Ep300 (Fig. 7C), which were mainly present at the PI3K-Akt signaling pathway and FoxO signaling pathway. In addition, the genes of Angptl4, Fabp4, Plin4 were obviously regulated by upstream transcription factor Pparg ( Figure. 7D), which were mainly present at the PPAR signaling pathway.

Discussion
This study has explored the therapeutic effect of WMP on memory impairment in AD mice and analyzed changes of gene expression in the hippocampus. Behavioral experiment illustreated that WMP could signi cantly improve the memory impairment of MOL mice, boosting spatial learning, memory, and recognition abilities. Hernandez-Rabaza et al. found apoptosis of hippocampus neurons was an important reason for the decline of learning and memory ability 20 . Slice analyses of hippocampus stained with Nestin (a marker of neural stem cells) showed the number of neurons were signi cantly decreased, which could be improved by WMP. Neural stem cells regulated nervous system homeostasis through self-renewal and multi-directional differentiation 21 . The hippocampus played an essential role in the process of learning and memory 22 . Since the neural stem cells in which could produce new neurons and glial cells to maintain the function of the hippocampus 23 . Previous studies had reported in ammation and oxidative stress were crucial risk factors for Alzheimer's disease, and the in ammatory factor occurred in pathologically vulnerable regions of AD 24 . Our results revealed an imbalanced brain redox in MOL mice, as well as the decreased oxidoreductase forming lipid peroxide (MDA) and activated in ammation factor (IL-6). Hence, WMP displayed a powerful ability to reduce oxidative stress and antiin ammation.
The hippocampus was a critical brain area for learning and memory in mammals, which took charge of short-term memories in daily life and speci c types of memories including episodic memory and spatial memory 25 ; 26 . In addition, the long-term enhancement (LTP) of the hippocampus has been recognized as the biological basis at the cell level of learning and memory activities 27 . Until now, this has been the rst study to uncover the changes of genes in the hippocampus under the in uence of WMP treatment in AD mice induced by D-galactose. Results have shown that WMP treatment regulated genes relevant to ribosomal, PI3K-Akt signaling pathway, FoxO signaling pathway, PPAR signaling pathway, neuroactive ligand-receptor interaction, and cellular senescence.
Ribosome played an important role in the process of protein translation and extension. The suspension of ribosomal would induce neuron apoptosis 28 ; 29 , and the translation level of new proteins was crucial for the learning and memory processing of neurons 30 . Ribosome transcription had priority in all cell transcription processes to adapt to ribosome production and protein synthesis, which was necessary to maintain long-term synaptic plasticity in the central nervous system 31 . Obvious ribosomal defects appeared in the Tua pathological area. The translation reduction caused by ribosome dysfunction has been considered as part of the AD process 32 . Our results showed WMP signi cantly regulated the ribosomal family genes (Rpl35a, Rps27rt, Rpl3l, Rpl21, Rpl26), and which genes strongly interacted.
Ribosomal family genes were the most promising targets, which involved in the rate controlling step of protein synthesis in the hippocampus. The experiment results found upstream transcription factor Ep300 obviously regulated genes of Cdkn1a, Spp1, Tnfsf10 in the hippocampus, which involved in in ammation and protein kinase. Upstream transcription factor Ep300 was a protein of aging-associated downregulation 33 , of which the suppression led to an increased expression of miR-142, as well as the accumulation of peroxisomes and ROS. MiR-142 was highly expressed in Alzheimer's disease, however, Ep300 would be able to inhibit the expression level of miR-142 [34][35][36] , which named Ep300 as an attractive target for AD therapeutic. Moreover, this study also revealed the remarkable regulated ability of the upstream transcription factor Pparg to genes of Angptl4, Fabp4, Plin4, which were mainly presented at PPAR (Peroxisome proliferator-activated receptors) signaling pathway. PPARγ were found to be able to restore the neural networks by stimulating the physiological Aβ clearance mechanism 37 . Angptl4 contributed to pathological vascular remodeling in capillary CAA, which could prevent the progression of Alzheimer's disease 38 . However, it is rst reported that Lipid metabolism related genes Fabp4 and Plin4 were closely related to Alzheimer's disease.

Declarations
Data availability The dataset generated and analyzed during the current study is available from the corresponding author on reasonable request.
Author contributions HL, DC, SZ designed the experiment, HL, DC, YM, GY and CX participated in the experimental study. HL analyzed the data and wrote the manuscript, DC, CC and SZ revised the manuscript. All authors contributed to the article and approved the submitted version.