Effect of WMP on behavioral experiment of Alzheimer's disease mice induced by D-galactose
Morris water maze is mainly reflecting 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 significantly restored the escape latency in MOL mice. On the fifth day, the platform was removed to conduct a positioning cruise test. The MOL mice took much longer time to find the hidden platform and stay less time in the target quadrant (Figure.1B-C). Both parameters could be significantly 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 significantly recovered after WMP treatment (Figure.1D). In the new object experiment, the recognition index of MOL mice was significantly 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 significantly decreased compared with CD group. Compared with MOL group, treatment with WMP could increase the brain index, spleen index, and liver index (Table 1).
Table 1: Effect of WMP on Organ Index of Alzheimer's disease mice induced by D-galactose. Columns indicated mean ± SD. Differences were expressed by ANOVA and denoted as follows: #p < 0.05, ##p < 0.01, ### p < 0.001, ####p < 0.0001vs. CD group; ∗ p < 0.05, ∗∗p < 0.01 vs. MOL group.
|
CD
|
MOL
|
WMP-L
|
WMP-H
|
Spleen index
|
9.992±2.466
|
4.910±0.890####
|
5.186±1.339
|
6.899±2.462*
|
Liver index
|
53.590±7.092
|
36.730±3.801####
|
40.850±3.229*
|
43.990±1.732***
|
Brain index
|
9.775±0.734
|
9.219±0.388#
|
9.497±0.356
|
9.788±0.752*
|
Heart index
|
5.137±0.853
|
4.487±0.322#
|
4.365±0.565
|
4.797±0.662
|
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 significantly 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 significantly 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 significantly 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 significantly 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 significant 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 significantly 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 significantly 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-specific 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 defined 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 significantly up-regulated transcripts (p < 0.05); the green dots correspond to significantly 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 down-regulated. 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 (4.35), H2-Q7 (3.44), H2-Q4 (2.17), Gzma (30.28), Pth2r (3.20), Npbwr1 (2.45), Col6a3 (0.35), Tnfsf10 (2.01), Cyp3a13 (0.46), Nanp (3.32), Rps27rt (4.33), Apol11b (8.99), Barx2 (2.56), Nxph4 (2.99), Scgn (0.30), Rny1 (0.08), and so on.
Table 2: Comparison of CD, MOL, and WMP cDNA library with the reference genome
Sample name
|
Total reads
|
Total mapped
|
Unique mapped
|
T-CD-1
|
52457516
|
50198445(95.69%)
|
47577061(90.70%)
|
T-CD-2
|
44929152
|
42981262(95.66%)
|
40929083(91.10%)
|
T-CD-3
|
50560398
|
48156331(95.25%)
|
45718057(90.42%)
|
T-MOL-1
|
43562928
|
41559866(95.40%)
|
39809885(91.38%)
|
T-MOL-2
|
54724000
|
52147214(95.29%)
|
49714687(90.85%)
|
T-MOL-3
|
50294482
|
48360406(96.15%)
|
45924448(91.31%)
|
T-WMP-1
|
45559774
|
43571375(95.64%)
|
41458266(91.00%)
|
T-WMP-2
|
41037328
|
39208853(95.54%)
|
37296676(90.88%)
|
T-WMP-3
|
44848818
|
42829451(95.50%)
|
40707412(90.77%)
|
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 classification 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 inflammatory 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 significant pathways in the hippocampus affected by WMP were related to immune, inflammatory 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 significantly up-regulated genes by treatment with WMP, pink circles correspond to significantly down-regulated 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.