Original title: "Sodium mannanate restrains the progression of Alzheimer's disease by therapeutic remodeling of intestinal microflora and inhibiting intestinal bacterial amino acid-like nerve infection".
1. Research background
It is generally believed that Alzheimer's disease is a neurodegenerative encephalopathy. At present, drugs for treating AD on the market can only improve symptoms, and no drugs can slow down or prevent the nerve damage caused by AD. In this paper, the researchers found that the intestinal flora may be related to Alzheimer's disease, so the changes of intestinal flora during AD and the mechanism of GV-97 1 were studied in a mouse model.
2. Method
2. 1 To study the changes of RNA level, intestinal flora and cells in 5XFAD transgenic (Tg) mice at different months.
2.2 Use antibiotics to remove intestinal flora, and determine the relationship between intestinal flora and neuroinflammation in the progress of AD.
2.3 In the behavioral experiment, the discrimination learning of Tg mice, WT mice and GV-97 1 treated mice was tested.
Changes of intestinal flora and cells in mice after GV-97 1 treatment.
2.5 The pathway enrichment analysis of mouse fecal metabolites was carried out.
2.6 To compare the differences of blood phenylalanine, isoleucine, Th 1 immune cells between AD patients with mild cognitive impairment and healthy people of the corresponding age.
3. Results
3. The progress of1ad is related to the changes of intestinal flora and the infiltration of immune cells.
The 5XFAD transgenic (Tg) mouse model was used to evaluate the role of intestinal flora changes in the pathogenesis of AD. The expression level of synaptophysin in hippocampus of Tg mice decreased significantly, indicating synaptic degeneration (figure 1a), while the behavior test of 9-month-old Tg mice showed a significant decrease in discriminating learning ability (figure 1b).
16SrRNA sequencing compared the intestinal types of Tg mice and WT mice in different stages of AD development. The analysis of intestinal types showed that Tg mice clustered in norank genus and WT mice clustered in Lactobacillus genus (Figure 1c). In TG mice, the intestinal microflora changed significantly during the development of AD, but hardly changed in WT mice (figure 1d). Using OTUs technology, it was found that the intestinal flora of Tg mice changed during AD (figure 1e). Immunohistochemical staining of IBA 1 (a sign of microglia activation) in brain slices of AD mice showed obvious microglia activation period (figure 1f). Microglial activation can be divided into two types: pro-inflammatory M 1 and neuroprotective M2 subtype. At the initial stage, both M 1 and M2 microglia increased, and then the subtype of M 1 increased continuously, reaching the peak in 7-9 months, while M2 microglia decreased from 3 months to 5 months, and then remained at a low level (Figure 1g). By analyzing the peripheral immune cells infiltrated in the brain during the development of AD, the frequency of cd45 high cells in the brain of Tg mice was significantly higher than that of WT mice, which was similar to IBA 1 staining (figure 1f, h). The changes of cd45 high cell subtypes at a series of time points during the progression of AD were further analyzed (Figure 1i). By k- means clustering, it was found that CD4+T cells were the main components of CD4 high cells, and the changes of M 1 cell were summarized (Figure 1i, J). Infiltrated Th 1 and Th2 cells are two main subtypes of CD4+ cells, showing similar dynamics to M 1 and M2 microglia (Figure 1k). With the change of intestinal flora pattern, the immune cell population tends to be dominated by Th 1 and M 1.
Figure 1. Intestinal microbial imbalance and changes of immune cells in TG mice during AD.
3.2 Intestinal flora is necessary for immune cell infiltration and microglia activation in the brain.
Antibiotic treatment in Tg mice resulted in a significant decrease in the number of microorganisms in the intestine (fig. 2a). With this change, the number of infiltrating pro-inflammatory Th 1 cell (Figure 2b) and M 1 cell (Figure 2c) in the brain decreased.
WT mice and Tg mice are raised together. The analysis of the changes of intestinal flora of these mice shows that the composition of intestinal flora of WT mice and Tg mice raised in the same way is very similar, but there are significant differences compared with WT mice raised alone (Figure 2d). In addition, the infiltration of Th 1 cells in * * * co-raised WT and Tg mice was also significantly higher than that in WT mice raised alone (Figure 2e). At the same time, the number of M 1 cells of co-bred WT mice only increased slightly (fig. 2f), indicating that the change of microbial population mainly affects infiltrating immune cells and non-resident immune cells. Consistent with the changes of immune cells, the expression of cytokines in the brain of WT and Tg mice showed obvious similarity, but WT mice were different (Figure 2g).
Figure 2. Intestinal flora is necessary for immune cell infiltration and microglia activation.
3.3 GV-97 1 can improve cognitive impairment.
In order to verify the important role of intestinal flora in the development of AD, the researchers treated APP/PS 1 mice with GV-97 1 (Figure 3a) for three months. Until 13 months old, GV-97 1 significantly improved the cognitive impairment, which indicated that the spatial learning ability and memory ability of APP/PS 1 mice were enhanced in Morris water maze (MWM) training test (Figure 3b) and probe test (Figure 3c). GV-97 1 can also significantly improve the performance of mice in y maze (fig. 3d).
Figure 3. Effect of GV-97 1 on behavior changes of APP/PS 1 mouse model.
3.4 GV-97 1 Alleviates neuroinflammation by changing intestinal flora.
Oral administration of GV-97 1 to Tg mice every month significantly changed the composition of intestinal flora (Figure 4a, B). Consistent with the changes of intestinal flora, Tg mice treated with GV-97 1 destroyed the correlation between the changes of brain lymphocytes and intestinal bacteria (Figure 4c), and the decrease of Th 1 cells in the brain (Figure 4d) significantly reduced the activation of microglia (Figure 4e) and the levels of various brain cell factors (Figure 4f). At the same time, GV-97 1 treatment significantly reduced the deposition of Aβ plaque and tau phosphorylation in Tg mice, and alleviated the decline of discriminating learning ability (Figure 4g–I).
Figure 4. GV-97 1 reduces neuroinflammation by readjusting intestinal flora.
3.5 GV-97 1 Inhibits neuroinflammation by regulating amino acid metabolism.
The pathway enrichment analysis of metabolites further revealed significant changes in amino acid-related metabolic pathways and enzymes, especially phenylalanine-related pathways (Figure 5a). Using random forest algorithm to classify these amino acids, phenylalanine is the most common between WT and Tg, followed by isoleucine, 5- hydroxytryptamine, histidine and acetylornithine (Figure 5b), indicating that they have a strong correlation with the progress of the disease. Then, the concentrations of the selected amino acids in feces and blood samples were examined in GV-97 1 treated or untreated Tg mice and compared with WT mice. The concentrations of phenylalanine and isoleucine in feces of Tg mice were significantly higher than those of WT mice, and GV-97 1 treatment significantly reduced their concentrations to a level comparable to that of WT mice (Figure 5c). The concentrations of phenylalanine and isoleucine in blood also changed similarly (Figure 5d). The differentiation of Th 1 cells exposed to phenylalanine or isoleucine was significantly enhanced, which was inhibited by GV-97 1 treatment (Figure 5e). Phenylalanine and isoleucine significantly promoted the proliferation of Th 1 cells, and GV-97 1 treatment inhibited the proliferation of Th 1 cells induced by phenylalanine, but the effect of isoleucine was almost unchanged (Figure 5f, blue column). In addition, the frequency of Th 1 cell in blood was significantly increased by injecting phenylalanine and isoleucine into wild-type mice (fig. 5g). The concentration of phenylalanine and isoleucine in blood and the frequency of Th 1 cell in patients with mild cognitive impairment were significantly higher than those in healthy subjects (fig. 5h, I). In addition, the elevated levels of phenylalanine and isoleucine in blood were also confirmed in MCI of another AD (Figure 5j), indicating that the abnormal accumulation of phenylalanine and isoleucine in blood and the increase of Th 1 frequency may be the characteristics of MCI of AD patients and MCI of healthy subjects.
Figure 5. GV-97 1 inhibits neuroinflammation by using amino acid metabolism.
conclusion
Figure 6. Schematic diagram of intestinal brain axis in the progress of AD and intervention strategies.
In addition to Aβ deposition and tau phosphorylation, the changes of intestinal flora during the development of AD will also cause metabolic disorders. Abnormal metabolites will cause peripheral inflammation, increase the brain infiltration of immune cells, and the immune cells will cross-talk with M 1 microglia in the brain, leading to pathological neuroinflammation and cognitive impairment (left figure). Oral administration of GV-97 1 can regulate intestinal flora, normalize disordered metabolites, reduce the infiltration of peripheral immune cells into the brain, solve neuroinflammation, reduce Aβ deposition and tau phosphorylation, and finally improve cognitive function (right).
GV-97 1 is an anti-AD drug, which has been proved to reverse the cognitive impairment of patients with mild to moderate AD in phase III clinical trials. This study found that GV-97 1 effectively restored the intestinal flora, reduced the concentrations of phenylalanine and isoleucine in feces and blood, and alleviated the cranial nerve inflammation related to Th 1. It is worth noting that Tg feces treated with GV-97 1 can simulate the curative effect of GV-97 1 to a great extent, while antibiotic treatment offsets the curative effect of GV-97 1. These findings indicate that the therapeutic effect of GV-97 1 is mainly achieved by reconstructing intestinal microflora. In this paper, the researchers believe that GV-97 1 provides an anti-AD strategy for microbial communities, which is worthy of further study.