3.1 FMT promoted body weight gain, enhanced demethylation of MeHg, increased fecal excretion and repaired tissue damage in MeHg-poisoned rats
The body weight gain since FMT in the 3 groups is shown in Fig. 1. It can be seen that the body weight gain in the MeHg group was lower than that in the Control group, suggesting that MeHg exposure affected the growth of rats. On the other hand, FMT effectively promoted the body weight gain from the 3rd day on, suggesting the health promotion capability of FMT in MeHg-poisoned rats.
Through H&E staining, inflammatory cell lesions were found in the intestinal tract and the hippocampal region of the brain, indicating serious intestinal and brain injuries in the MeHg group[28–29] (Fig. 2). On the other hand, in the MeHg + FMT group, the inflammatory cell lesions in the intestinal and brain injuries disappeared. FMT also relived the damage to other organs, such as decreased number of inflammatory cells in the liver and spleen, which can be seen in Figure S1. It has been reported that FMT has a certain recovery effect on enteritis[41], liver injury[42], kidney injury[43] and brain nerve injury [44].
THg and MeHg levels in different tissues with or without FMT are shown in Fig. 3. The order of THg from high to low was kidney > liver > spleen > brain > gastrointestinal tissues in the MeHg group. With FMT treatment, the levels of THg decreased significantly, especially in the liver, kidney and brain in the MeHg + FMT group (Fig. 3A). For example, after FMT treatment, the THg concentration in the liver was reduced from 250 ± 21 to 50 ± 12 µg/g, in the kidney from 300 ± 34 to 175 ± 16 µg/g, and in the brain from 100 ± 11 to 50 ± 15 µg/g, respectively.
The levels of MeHg were much lower than THg in different tissues, suggesting the demethylation of MeHg. FMT was also found to reduce the levels of MeHg in different tissues. For example, after FMT treatment, the MeHg concentration in the liver was reduced from 80 ± 9 to 20 ± 3 µg/g, in the kidney from 100 ± 14 to 30 ± 4 µg/g, and in the brain from 50 ± 5 to 15 ± 2 µg/g, respectively.
Furthermore, the MeHg/THg with or without FMT was calculated. The proportions of MeHg/THg in liver, kidney, brain, spleen, stomach, small intestine and large intestine were 32%, 33.3%, 50%, 40%, 40%, 80% and 40% in the MeHg group, while those in MeHg + FMT group were: 20%, 17.1%, 20%, 16%, 60% and 40%, respectively, indicating that FMT increased the demethylation rate.
3.2 FMT reconstructed gut microbiota in MeHg-poisoned rats
Figure 4 shows the changes of the gut microbiota in MeHg-poisoned rats with or without FMT. Operational taxonomic unit (OTU) was used to classify the gut microbiota as shown in Fig. 4A. Compared with the control group, there were 8,449 similar OTUs in the MeHg group and 9,299 similar OTUs in the MeHg + FMT group, indicating that FMT improved the disturbance effect of MeHg on the intestinal microbial community.
UPGMA (Unweighted pair group method with arithmetic mean) assumes that all nucleotides/amino acids are with the same mutation rate. The evolutionary distance between samples can be observed through the distance of branches and the distance of clusters. In Fig. 4B, it can be seen that the distance of branches of MeHg + FMT groups was closer to the Control group than the MeHg group, suggesting that FMT restored intestinal microbial disturbance caused by MeHg.
Figure 4C, D shows α/β diversity index between groups. α diversity index can reflect the species richness, β diversity index can reflect the habitat diversity. α diversity index shanon was in Fig. 4C, the box chart reflects the median, dispersion, maximum, minimum and outlier of species diversity among different groups, it can be seen that the FMT group and the Control group showed a difference index, while the MeHg group did show a similar index, indicating that the FMT group and the Control group had a certain normal α diversity. β diversity index PcoA was presented in Fig. 6D, there were some similarities and differences in the three groups, and the differences were shown as differences in the dimensions of PcoA1(18.66%) and PcoA2(12.56%), indicating that the groups showed normal habitat diversity.
Figure 4E shows the composition of the gut microbiota at the phylum level. Compared with the control group, MeHg down regulated Firmicutes, while up regulated Bacteroidetes and Proteobacteria, which were the dominated bacteria in intestine(P < 0.05)[54]. On the contrary, the change the abundance of Firmicutes were was consistent with that of control group, while the abundance up of Bacteroidetes and Proteobacteria induce by MeHg were restored by FMT(P > 0.05). The above results indicate that FMT can repair the intestinal microbial disturbance caused by MeHg at the family level.
Figure 4F shows the abundance of gut microbiota at the family level (VS Control > 2). Compared to the control group, MeHg up regulated the relative abundance of Succinivibrionaceae(18 fold change), Rikenellaceae(4 fold change), Christensenellaceae(4 fold change), Streptococcaceae(4 fold change), Bacteroidaceae(4 fold change), Uncultured bacterium(2 fold change), Eggerthellaceae(2 fold change), while down regulated of Enterobacteriaceae(-22 fold change), Lactobacillaceae(-4 fold change), Spirochaetaceae(-1 fold change), Peptostreptococcaceae(-2 fold change), Enterococcaceae(-22 fold change). FMT can effectively restore intestinal microbial disturbance caused by MeHg, such as Succinivibrionaceae(4 fold change), Rikenellaceae(2 fold change), Christensenellaceae(2 fold change), Streptococcaceae(1 fold change), Bacteroidaceae(1 fold change), Uncultured bacterium(1 fold change), Eggerthellaceae(-2 fold change), Enterobacteriaceae(-16 fold change), Lactobacillaceae(-1 fold change), Spirochaetaceae(-8 fold change), Peptostreptococcaceae(-12 fold change), Enterococcaceae(-8 fold change).
The following microbial changes have been reported to be closely related to the activation of the gut-brain axis system. Bacteroidaceae[55], Rikenellaceae[56], Streptococcaceae[57], Lactobacillaceae[58]. MeHg up regulated the relative abundance of Bacteroidaceae, Rikenellaceae, Streptococcaceae, while down regulated, Lactobacillaceae. Showing that MeHg activates the gut-brain axis system. FMT can effectively restore intestinal microbial disturbance caused by MeHg, showed that FMT could reverse changes in enterobrain axis-related bacterial abundance.
In addition, sulfate-reducing bacteria have been reported to effectively regulate the demethylation of MeHg in the intestine[59]. MeHg demethylation is associated with bacteria such as Desulforibrionaceae(6 fold change), Clostridiaceae 1(-14 fold change) have significant changes caused by MeHg, suggesting that these gut microbiota promote MeHg demethylation, and FMT induce the Desulforibrionaceae(3 fold change), Clostridiaceae 1(-26 fold change), the results showed that FMT promoted demethylation of MeHg. More detailed information on changes at the family level is shown in Table S1.
3.3 FMT reversed the changes of gut-brain axis related metabolites and other biomarkers in MeHg-poisoned rats
The metabolome changes (volcano map, PCA analysis, cluster analysis, specific differential metabolites) in fresh feces in MeHg and MeHg + FMT group and the Control group are presented in Fig. 5.
Figure 5A shows that MeHg exposure led to 4616 metabolites with 270 metabolites (144 increased and 126 decreased, > 1.5 folds p < 0.05) are statistically different compared to the Control group, and 4346 similar metabolites. Figure 4B shows that FMT led to 5310 metabolites with 368 statistically significant (152 increased and 216 decreased, > 1.5 folds, p < 0.05) are statistically different compared to the Control group, and 4942 similar metabolites. The number of similar OTU in FMT group compared with Control group was higher than that in MeHg group compared with Control group. These results suggested that the metabolic disorder caused by MeHg was improved after FMT.
The PCA results is shown in Fig. 5C. The MeHg + FMT group is closer in spatial position to the MeHg and Control groups, indicating that FMT has a certain recovery effect on MeHg induced metabolic disorder.
Cluster analysis of differential metabolites using stratified cluster heat maps constructed with 2-fold variation of molecular characteristics (p < 0.05) (Fig. 4D). The color blocks of the MeHg group had a significant difference compared to the Control group, while the MeHg + FMT group was similar to that of the Control group. It indicating that FMT has a certain recovery effect on MeHg induced metabolic disorder.
The metabolites disrupted in MeHg and MeHg + FMT group are shown in Fig. 4E. MeHg mainly caused changes of metabolites related to nerve especially gut-brain axis related ones, such as down-regulated of L-Tyrosine[45], Glycine[46], Aspartic acid[47], Leucine[48], up-regulated of GABA[50], Serine[49], Glutamate[45] and Xanthemnic acid[51], which is consistent with our previous results[28–29]. On the other hand, FMT up-regulated L-Tyrosine, Glycine, Aspartic acid, Leucine and, up-regulated of GABA, Serine, Glutamate and Xanthemnic acid. More detailed information on the metabolite changes are shown in Table S2. It indicates that FMT modulates the gut-brain axis related metabolites.
BDNF is an important nutrient factor of brain nerves, which regulates the survival, growth, differentiation and apoptosis of nerve cells, and may protect neurons from stress by affecting the regeneration of nerve cells in the hippocampus and synaptic plasticity[52]. It is also a mediator involved in the survival of neurons, as well as the plasticity of dopamine, 5-hydroxytryptamine and cholinergic neurons in the central nervous system, and is a molecular marker of neuroplasticity. The levels of BDNF in serum, gut and brain are shown in Fig. 6A/B/C. It was found that MeHg significantly decreased the level of BDNF in serum, brain and gut compared to the control group as shown in Fig. 6A/B/C (p < 0.001), while FMT reversed the levels of BDNF in serum, brain, and intestinal levels. It indicated that MeHg disturbed the secretion of BDNF in the intestine and brain due to the damage to intestinal and brain structures.These results indicated that fecal transplantation could regulate BDNF and treat MeHg neurotoxicity.
50% of dopamine in the human body is secreted by the gut, so the decrease of dopamine secretion in the gut directly affects the whole body's dopamine metabolism[53]. The discovery that MeHg can induce the secretion of dopamine in the gut, brain and serum proves that MeHg does affect the gut brain axis system, and fecal transplantation can indeed repair such changes. The levels of dopamine in serum, gut and brain are shown in Fig. 6D/E/F. It was found that MeHg significantly decreased the level of dopamine in serum, brain and gut compared to the control group as shown in Fig. 6D/E/F (p < 0.001), while fecal transplantation reverses MeHg-induced dopamine reduction in serum, brain, and gut levels. It indicated that MeHg disturbed the secretion of dopamine in the intestine and brain due to the damage to intestinal and brain structures.These results indicated that fecal transplantation could regulate dopamine and treat MeHg neurotoxicity.