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Sleep deprivation (SD) disrupts the lives of millions of people worldwide and profoundly affects cognitive and physical function. Individuals with acute SD are at increased risk of Alzheimer's disease and cardiovascular disease, with increased levels of systemic inflammation. SD affects the whole body, involving systemic damage to multiple tissues. However, the underlying mechanism by which SD causes cognitive impairment remains unclear.
On January 31, 2023, the team of Professor Chen Yaoxing from the School of Veterinary Medicine of China Agricultural University published an article entitled "Gut microbiota-derived metabolites mediate the neuroprotective Effect of melatonin in cognitive impairment induced by sleep deprivation ("Gut flora and its metabolites mediate the neuroprotective effect of melatonin on sleep deprivation-induced cognitive impairment)". This paper uses flora transplantation experiments, Aeromonas colonization and LPS or butyrate supplementation assays, assessing the mediation of gut microbiota and their metabolites in melatonin-improved cognitive impairment in sleep-deprived mice, revealed that melatonin (Mel) Improving the effect and mechanism of sleep deprivation-induced cognitive impairment. BMEC provided full-length microbial diversity sequencing and non-target metabolome detection services for this study .
material method
Materials: Male ICR mice, BV2 cell line, collected mouse feces, colon contents, hippocampal tissue
Methods: Full-length 16s rDNA sequencing of colon contents, non-target metabolome detection, SCFA detection, hippocampal Western blot, ELISA, qPCR
Experimental Design: Microflora Transplantation, Aeromonas Colonization, LPS Supplementation, Butyrate Supplementation Trial
Figure 1. Schematic and timeline of the experimental model.
A: Fecal microbiota transplantation (FMT) experiment. B: Fixed value experiment of Aeromonas veronii.
C: LPS (lipopolysaccharide) treatment experiment. D Butyrate treatment experiment.
Mel: Melatonin, MWM: Morris water maze, Vehicle: 2% ethanol sterile saline. SD: sleep deprivation, TAK-242: TLR4 inhibitor
Research technology route
main results
1. Gut microbiota mediates the neuroprotective effect of melatonin on SD-induced neuroinflammation and memory impairment
To test whether the neuroprotective effect of Mel (melatonin) on SD-induced memory impairment depends on the gut microbiota, a fecal microbiota transplantation experiment (FMT) was performed. Fecal microbiota from CON, SD, or SD + Mel groups were transplanted into gut microbiota-depleted mice for 2 weeks (Fig. 2A). After the addition of antibiotics, the bacteria in the feces of the mice were greatly reduced (Fig. 1B). Behavioral results showed that mice in the SD-FMT group had a 115.8% higher latency to reach the platform than the CON-FMT group, and had a 143.3% longer path length in the first test (using the hidden platform) (Fig. 1C, EF). Pathway efficiencies were not significantly different between groups (Fig. 2G). In the second test without a hidden platform, the SD-FMT group entered the target area 55.2% less often and spent 36.4% less time than the CON-FMT group, with no difference between the SD + Mel-FMT and CON-FMT groups Significant differences (Fig. 2D, H, I). Memory impairment was improved in mice carrying SD+Mel donor microbiota compared with SD donors, suggesting that gut microbiota may contribute to Mel conferring cognitive dysfunction benefits in SD mice (Fig. 2C–I ).
To investigate whether SD-mediated gut microbiota induces neuroinflammation, changes in inflammatory cytokines and microglia immunohistochemical staining in the hippocampus were further evaluated. The cumulative optical density (IOD) of Iba1-positive cells in hippocampus CA1, CA3 and dentate gyrus (DG) in SD-FMT group was 51.9%, 27.6% and 32.3% higher than that in Con-FMT group, respectively. It was observed that the levels of IL-6 and TNF-α in the hippocampus of SD-FMT mice were significantly increased, while the levels of IL-4 and IL-10 were significantly decreased). However, FMT of "SD+MEL microbiota" significantly inhibited the activation of microglia, increased pro-inflammatory factors, and decreased anti-inflammatory factors. The expression levels of cleaved caspase-3, Bax and Bcl-2 were further detected by immunoblotting. Compared with the CON-FMT group, the expression of Bcl-2 was significantly down-regulated, the expression of cleaved caspase-3 was significantly up-regulated, and the expression of bax was significantly up-regulated in the SD-FMT group. However, FMT of the "SD+MEL microbiota" reversed these changes. FMT experiments showed that the protective effect of Mel against SD-induced neuroinflammation, apoptosis, and memory impairment requires gut microbiota.
Figure 2. Gut microbiota mediates the neuroprotective effect of melatonin against sleep deprivation-induced memory impairment
2. Effect of FMT treatment on the intestinal flora composition of recipient mice
To verify whether FMT regulates the gut microbiota, full-length 16S rDNA gene sequencing was performed to analyze the bacterial taxonomic composition of FMT in recipient mice. A total of 21 samples were obtained from three groups of mice (n=7) and subsequently sequenced to generate V1-V9 16S rRNA gene profiles. The results showed that there were no significant differences in CHO1, ACE, Simpson and Shannon indexes among the three groups. PCoA analysis showed that PC1, PC2 and PC3 explained 17.96%, 15.50% and 9.66% of the variance, respectively.
Figure 3. Composition of the colonic microbiota of FMT-treated mice
LEfSe analysis indicated that Bacteroidetes and Proteobacteria were more abundant in the SD-FMT group than in the CON-FMT and SD+Mel-FMT groups. Furthermore, Firmicutes were decreased in the SD-FMT group compared with the CON-FMT and SD+Mel-FMT groups. Using LEfSe analysis, 64 taxa biomarkers were identified in the three groups. The relative abundance of lachnospiraceae_NK4A136_group, Eubacteriumxylanophilum_group, Ruminococcus_1 and Lachnospiraceae_A2 in SD-FMT group was significantly lower than that in CON-FMT and SD + Mel+FMT groups, and the relative abundance of Turicimonas in SD-FMT group was significantly higher than that in CON-FMT and SD + Mel-FMT group, while there was no significant difference between CON-FMT and SD + Mel-FMT groups.
Figure 4. Composition and key microbiota of the colonic microbiota of FMT-treated mice.
3. Effect of FMT treatment on the composition of metabolites of intestinal flora of recipient mice
Metabolic profiling was further performed on FMT recipient mice. Analysis of metabolites revealed 2260 metabolites in the colon. Venn diagrams showed that different treatments resulted in different metabolite changes (Fig. 5A). Principal component analysis showed that the metabolite composition of the microbiota in all groups was significantly clustered, closer to the CON-FMT group than the SD-FMT group (Fig. 5B). In order to further verify the differences between samples in different groups, OPLS-DA analysis was carried out, and the OPLS-DA model showed a good separation among the three groups. Volcano plots demonstrate up- and down-regulation of differential metabolites (p<0.05, |log2FC|>1) (Fig. 4E–G). Compared with the CON-FMT group, 547 metabolites were upregulated and 15 metabolites were downregulated in the SD-FMT group. Compared with the SD-FMT group, 574 metabolites increased and 26 metabolites decreased in the SD+MEL-FMT group. In addition, 41 most varied metabolites among the three groups were screened out (Fig. 5J). Compared with the SD-FMT group, the contents of butyric acid (butyric acid) and L-tryptophan (L-tryptophan) in the SD+Mel-FMT group were significantly increased.
Figure 5. Composition of colonic microbiota metabolites in FMT-treated mice
4. Correlations between microbiome composition and phenotypic variables
In order to further investigate whether the metabolites were changed, the content of short-chain fatty acids in feces was determined by GC/MS. The results showed that the fecal butyrate content of SD-FMT group was significantly lower than that of Con-FMT group. Butyric acid content was significantly increased in the SD+MEL-FMT group compared with the SD-FMT group. The relative abundance of Aeromonas in the colon was determined by RT-PCR, and the level of LPS in the hippocampus was determined by ELISA. The LPS and Aeromonas in the SD-FMT group were significantly higher than those in the Con-FMT group. SD+MEL-FMT group was significantly lower than SD-FMT group.
Correlation analysis showed that the relative abundance of Aeromonas was positively correlated with LPS levels. Fecal butyrate levels were positively correlated with microorganisms g_lachnospiraceae_NK4A136_group, g_eubia_xylanophilum, g_lachnospiraceae_A2 , and s_CIEAF020 , and negatively correlated with g_Turicimonas and s_Turicimonas_Muris . These findings suggest that changes in these microbes may be associated with changes in fecal butyrate levels (Fig. 6A). The microbial g_Lachnospiraceae_NK4A136_group was negatively correlated with latency to reach the platform, path length to reach the platform, TNF-α, IL-6, and LPS, and positively correlated with time to enter the target area, number of entries, and IL-10. Therefore, the gut microbe-mediated effects of SD or Mel treatment groups may be related to their potential to regulate microbial metabolites (LPS and butyrate).
Figure 6. Correlations between microbiome composition and phenotypic variables.
5. Melatonin attenuates the occurrence of neuroinflammation and memory impairment in mice induced by A. veronii colonization
To verify the role of elevated levels of Aeromonas in SD-induced memory impairment, the researchers constructed a mouse model colonized with Aeromonas (Fig. 7A) . After Veronii colonization, increases in latency and path length for mice to reach the platform were observed, whereas time and number of entries in the mouse target area were observed. In addition, the cumulative absorbance of Iba1-positive cells in the hippocampal CA1, CA3 and DG regions of the rats in the Aero group was 23.1%, 23.9% and 22.4% higher than that in the CON group, respectively. Compared with the CON group, the levels of LPS, IL-6 and tumor necrosis factor-α in the hippocampus of rats in the AERO group were significantly increased, the levels of IL-4 and IL-10 were significantly decreased, and the expression level of TLR4 was increased. There was a significant difference between the Aero group and the CON group. However, supplementation with Mel inhibited this process, resulting in no significant difference between the CON and A+Mel groups.
Figure 7. Melatonin ameliorates the development of neuroinflammation and memory impairment induced by Aeromonas colonization in mice.
6. The effect of butyrate on melatonin improves memory impairment in SD mice
To investigate whether butyrate could mediate the improved SD-induced cognitive impairment caused by Mel, the CON, SD, SD + Abs, SD + Mel, SD + Abs + Mel and SD + Abs + butyrate groups were observed Changes in the MWM (Morris water maze) test when mice were placed on a hidden or visible platform (Fig. 8A). Compared with the CON group, the SD group and the SD+Abs group had obvious spatial memory impairment. The latter two groups showed a significant increase in path length and latency to reach the platform, and a significant decrease in the time spent and number of entries in the destination area. In contrast, Mel supplementation reversed SD-induced changes in spatial memory impairment; in path length and delay to reach platform, time spent, and target area between SD + Mel, SD + Abs + Mel, and CON groups No significant difference was observed in the number of entries. Similar to Mel supplementation, butyrate supplementation improved SD-induced cognitive impairment. No differences were observed in any parameter between the SD+Abs+butyrate and CON groups (Fig. 8B–H). Furthermore, there was no significant difference in path efficiency between the groups. Thus, the results of the butyrate treatment experiments suggest that butyrate, as a signaling molecule of the gut microbiota, may mediate the improvement of Mel on SD-induced cognitive impairment.
To investigate whether butyrate could mediate the improvement of SD-induced neuroinflammation and apoptosis by Mel, changes in Iba1 expression and release of inflammatory cytokines and intracellular signaling proteins in the hippocampus were examined. Compared with the CON group, neuroinflammation was evident in the SD and SD + Abs groups, which indicated a significant increase in the IOD of Iba1-positive cells in hippocampal CA1, CA3 and DG, IL-6 and TNF-α levels, and IL-4 and IL- 10 levels are significantly lower. Compared with the CON group, the expressions of intracellular signaling proteins HDAC3, p-IκB, p-P65, and cleaved caspase-3 were significantly upregulated in the SD and SD + Abs groups; on the contrary, Mel supplementation reversed the neuroinflammation and apoptosis induced by SD death changes. The above indicators showed no significant difference among SD + Mel, SD + Abs + Mel and CON groups. Similar to Mel supplementation, butyrate supplementation also ameliorated SD-induced neuroinflammation and apoptosis. No differences were observed in any parameter between the SD+Abs+butyrate and CON groups.
Figure 8. Melatonin improves lipopolysaccharide-induced neuroinflammation and memory impairment in mice
7. Effect of butyrate on LPS-induced inflammatory response and neurotoxicity of BV2 cells
To explore the role of LPS or butyrate metabolites in SD-induced Mel-induced cognitive impairment, neuroinflammation was simulated with LPS and butyrate was used as an intervention. As expected, exposure of BV2 cells to LPS resulted in increased tumor TNF-α and IL-6 secretion and decreased IL-4 and IL-10 secretion. After LPS treatment, the protein expression levels of HDAC3, p-iκB and p-p65 in BV2 cells increased.
In addition, a microglia conditioned medium (CM) system was used to assess whether butyrate attenuation of microglial neurotoxicity was associated with neuronal survival. CM extracted from LPS-induced BV2 microglia were added to HT22 cells treated with or without butyrate. Lipopolysaccharide-induced CM stimulates apoptosis of HT22 cells by upregulating cleaved caspase-3 levels. However, butyrate effectively reversed these LPS-induced changes. In contrast, after TAK242 treatment, HDAC3, p-iκB and p-p65 proteins were down-regulated compared with LPS group. The results showed that the inhibition of MCT1 by AZD3965 led to up-regulation of HDAC3, p-iκB and p-p65 proteins in the LPS+butyrate+AZD3965 treatment group compared with the LPS treatment group. In addition, compared with the LPS+butyrate group, the protein expression of p-IκB and p-p65 was up-regulated after treatment with ITSA-1 as HDAC3 agonist, and had no significant effect on the expression level of HDAC3 protein. However, treatment with pDTC (an NF-κB antagonist) had similar beneficial effects as butyrate.
Figure 9. Experimental study of butyric acid improving memory impairment in sleep deprived mice
Summarize
This study reveals the protective effect of Mel on cognitive impairment caused by SD. Further mechanistic studies indicated that the down-regulation of Aeromonas and lipopolysaccharide (LPS) levels and the up-regulation of Lachnospiraceae NK4A136 and butyrate levels may be the underlying mechanism of Mel exerting neuroprotective effects in SD-induced cognitive impairment.
references
Wang X, Wang Z, Cao J, Dong Y, Chen Y. Gut microbiota-derived metabolites mediate the neuroprotective effect of melatonin in cognitive impairment induced by sleep deprivation. Microbiome. 2023;11(1):17. Published 2023 Jan 31. doi:10.1186/s40168-022-01452-3
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