Gut microbiota-derived metabolites mediate the neuroprotective effect of melatonin in cognitive impairment induced by sleep deprivation

Abstract

Sleep loss is a serious global health concern. Consequences include memory deficits and gastrointestinal dysfunction. Our previous research showed that melatonin can effectively improve cognitive impairment and intestinal microbiota disturbances caused by sleep deprivation (SD). The present study further explored the mechanism by which exogenous melatonin prevents SD-induced cognitive impairments. Here, we established fecal microbiota transplantation, Aeromonas colonization and LPS or butyrate supplementation tests to evaluate the role of the intestinal microbiota and its metabolites in melatonin in alleviating SD-induced memory impairment. RESULTS: Transplantation of the SD-gut microbiota into normal mice induced microglia overactivation and neuronal apoptosis in the hippocampus, cognitive decline, and colonic microbiota disorder, manifesting as increased levels of Aeromonas and LPS and decreased levels of Lachnospiraceae_NK4A136 and butyrate. All these events were reversed with the transplantation of SD + melatonin-gut microbiota. Colonization with Aeromonas and the addition of LPS produced an inflammatory response in the hippocampus and spatial memory impairment in mice. These changes were reversed by supplementation with melatonin, accompanied by decreased levels of Aeromonas and LPS. Butyrate administration to sleep-deprived mice restored inflammatory responses and memory impairment. In vitro, LPS supplementation caused an inflammatory response in BV2 cells, which was improved by butyrate supplementation. This ameliorative effect of butyrate was blocked by pretreatment with MCT1 inhibitor and HDAC3 agonist but was mimicked by TLR4 and p-P65 antagonists. CONCLUSIONS: Gut microbes and their metabolites mediate the ameliorative effects of melatonin on SD-induced cognitive impairment. A feasible mechanism is that melatonin downregulates the levels of Aeromonas and constituent LPS and upregulates the levels of Lachnospiraceae_NK4A136 and butyrate in the colon. These changes lessen the inflammatory response and neuronal apoptosis in the hippocampus through crosstalk between the TLR4/NF-κB and MCT1/ HDAC3 signaling pathways. Video Abstract.

Keywords: Cognitive impairment; Hippocampus; Melatonin; Microbial–gut–brain axis; Sleep deprivation.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2023. The Author(s).

Figures

Fig. 1

Schematic and timeline of the experimental model. A Fecal microbiota transplantation (FMT) experiment. BAeromonas veronii colonization experiment. C LPS treatment experiment. D Butyrate treatment experiment. Mel: melatonin, MWM: Morris water maze, Vehicle: 2% ethanol sterile saline. SD: sleep deprivation, TAK-242: TLR4. inhibitor

Fig. 2

The gut microbiota mediated the neuroprotective effect of melatonin in memory impairment induced by sleep deprivation. A Schematic illustration of experimental design. B Comparison of bacterial colony-forming unit (CFU) in feces from control- and Abs-treated mice (n = 10). C Track plot of spatial memory test (with hidden platform). D Track plot of spatial memory test (without hidden platform). E Latency to reach the platform (n = 8). F Path length to reach the platform (n = 8). G Path efficiency to reach the platform (n = 8). H Time spent in the target zone (n = 8). I Number of entries into the target zone (n = 8). J Images of the immunohistochemical microglia in the different experimental groups. The immunohistochemical results were processed using ImageJ. Bar = 50 μm. K IOD of Iba1-positive cells in the hippocampal cornu ammonis (CA)1, CA3, and dentate gyrus (DG) regions (n = 6). L–O The levels of cytokines (TNF-α, IL-6, IL-4, and IL-10) in the hippocampus (n = 5). CON-FMT: receiving control microbiota FMT mice, SD-FMT: receiving sleep deprivation microbiota FMT mice, SD + Mel-FMT: receiving SD + Mel (20 mg/kg) microbiota FMT mice, V-FMT: receiving vehicle microbiota FMT mice. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 3

Composition of the colonic microbiota in FMT-treated mice. A,B Alpha diversity evaluation of colon microbial richness and evenness by measuring Chao and Ace diversity indexes. C Principal component analysis (PCA). D PCoA score plot. E Nonmetric multidimensional scaling (NMDS) score plot based on the binary_jaccard distance plot based on the OTU of the gut microbe. F Shannon curves. G OTU rank curves. H Rarefaction curves. I Rank abundance curve. J Unweighted pair-group method with arithmetic mean (UPGMA) analysis (at the phylum level). K Unweighted pair-group method with arithmetic mean (UPGMA) analysis (at the genus level) in the mice cecum of the CON-FMT, SD-FMT, and SD + Mel-FMT groups. CON-FMT: receiving control microbiota FMT mice, SD-FMT: receiving sleep deprivation microbiota FMT mice, SD + Mel-FMT: receiving SD + Mel (20 mg/kg) microbiota FMT mice. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

Composition and the key microflora of the colonic microbiota in FMT-treated mice. A Relative abundances of colonic microbiota at the phylum level in the 3 groups. B Relative abundances of gut microbiota at the genus level in the 3 groups. C Linear discriminant analysis effect size (LEfSe) was performed to identify the bacteria that are differentially represented between the different groups. D–K Relative abundance of p_Bacteroidota, p_Proteobacteria, c_Gammaproteobacteria, g_Lachnospiraceae_NK4A136, g_Eubacterium_xylanophilum, g_Ruminococcus_1, g_ Lachnospiraceae_A2, and g_Turicionas in the colon microbiota based on the LefSe results. Solid and dashed lines indicate the mean and median, respectively. CON-FMT: receiving control microbiota FMT mice, SD-FMT: receiving sleep deprivation microbiota FMT mice, SD + Mel-FMT: receiving SD + Mel (20 mg/kg) microbiota FMT mice

Fig. 5

Composition of the colonic microbiota metabolites in FMT-treated mice. A Venn based on the microbiota metabolites. B β-diversity of principal component analysis (PCA). C Orthogonal projections to latent structures-discriminate analysis (OPLS-DA) score plot was performed on colon. D In the permutation validation plot the Y-axis intercepts of R2 and Q2 are 0.587 and 0.273, respectively, indicating that the model is valid. E Volcano plot based on the differential metabolite screening compared with the CON-FMT and SD-FMT groups. F volcano plot based on the differential metabolite screening compared with the CON-FMT and SD + Mel-FMT groups. G volcano plot based on the differential metabolite screening compared with the SD-FMT and SD-FMT groups. H The relative abundance of butyric acid. I The relative abundance of L-Tryptophan. J Heatmap showing the relative abundance of the key identified 41 metabolites (p < 0.05, VIP > 1). CON-FMT: receiving control microbiota FMT mice, SD-FMT: receiving sleep deprivation microbiota FMT mice, SD + Mel-FMT: receiving SD + Mel (20 mg/kg) microbiota FMT mice. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

Correlation between the microbiome composition and phenotypic variables. A The correlation of top focus metabolites and the signature microbiota (Spearman correlation test, FDR < 0.05). B The contents of acetate in the feces (n = 8). C The contents of propionate in the feces (n = 8). D The contents of butyrate in the feces (n = 8). E The levels of LPS in the hippocampus (n = 7). F Relative abundance of colonic Aeromonas (n = 7). G Plots of correlation analysis between the fecal level of butyrate and colonic Aeromonas. X-axis represented the Relative abundance of colonic Aeromonas; Y-axis represented the fecal butyrate levels. CON-FMT: receiving control microbiota FMT mice, SD-FMT: receiving sleep deprivation microbiota FMT mice, SD + Mel-FMT: receiving SD + Mel (20 mg/kg) microbiota FMT mice. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

Melatonin ameliorates the occurrence of neuroinflammation and memory impairment in mice induced by Aeromonas colonization. A Schematic illustration of experimental design. B Track plot of spatial memory test (with hidden platform). C Track plot of spatial memory test (without hidden platform). D Latency to reach the platform (n = 8). E Path length to reach the platform (n = 8). F Time spent in the target zone (n = 8). G Number of entries into the target zone (n = 8). H Path efficiency to reach the platform (n = 8). I The levels of LPS in the hippocampus (n = 7). J–M The levels of cytokines (TNF-α, IL-6, IL-4, and IL-10) in the hippocampus (n = 5). N Images of the immunohistochemical microglia in the different experimental groups. The immunohistochemical results were processed using ImageJ. Bar = 50 μm. O IOD of Iba1-positive cells in the hippocampal cornu ammonis (CA)1, CA3, and dentate gyrus (DG) regions (n = 6). P–T Relative protein levels of TLR4, HDAC3, p-IκB, p-P65, and cleaved caspase-3 in the hippocampus (n = 6). CON: control group, Aero: Aeromonas colonization group, A + Mel: Aeromonas + melatonin (20 mg/kg) group. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 8

Melatonin ameliorates LPS-induced neuroinflammation and memory impairment in mice. A Schematic illustration of experimental design. B Track plot of spatial memory test (with hidden platform). C Latency to reach the platform (n = 8). D Path length to reach the platform (n = 8). E Path efficiency to reach the platform (n = 8). F Track plot of spatial memory test (without hidden platform). G Time spent in the target zone (n = 8). H Number of entries into the target zone (n = 8). I Images of the immunohistochemical microglia in the different experimental groups. The immunohistochemical results were processed using ImageJ. Bar = 50 μm. J The levels of LPS in the hippocampus (n = 7). K IOD of Iba1-positive cells in the hippocampal cornu ammonis (CA)1, CA3, and dentate gyrus (DG) regions (n = 6). L–O The levels of cytokines (TNF-α, IL-6, IL-4, and IL-10) in the hippocampus (n = 5). P–T Relative protein levels of TLR4, HDAC3, p-IκB, p-P65, and cleaved caspase-3 in the hippocampus (n = 6). CON: control group, LPS: lipopolysaccharides (2 mg/kg) group, LPS + Mel: LPS + melatonin (20 mg/kg) group, LPS + TAK-242: LPS + TAK-242 (TLR4 inhibitor, 150 mg/kg) group. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 9

Effect of butyrate on Mel improved memory impairment in sleep-deprived mice. A Schematic illustration of experimental design. B Track plot of spatial memory test (with hidden platform). C Latency to reach the platform (n = 8). D Path length to reach the platform (n = 8). E Path efficiency to reach the platform (n = 8). F Track plot of spatial memory test (without hidden platform). G Number of entries into the target zone (n = 8). H Time spent in the target zone (n = 8). I Images of the immunohistochemical microglia in the different experimental groups. The immunohistochemical results were processed using ImageJ. Bar = 50 μm. J–L IOD of Iba1-positive cells in the hippocampal cornu ammonis (CA)1, CA3, and dentate gyrus (DG) regions (n = 6). M–P The levels of cytokines (TNF-α, IL-6, IL-4, and IL-10) in the hippocampus (n = 5). Q–T Relative protein levels of HDAC3, p-IκB, p-P65, and cleaved caspase-3 in the hippocampus (n = 6). CON: control group, SD: sleep deprivation group, SD + Mel: SD + melatonin (20 mg/kg) group, SD + Abs: SD + antibiotics group, SD + Abs + Mel: SD + antibiotics + Mel group, SD + Abs + Butyrate: SD + antibiotics + Butyrate (40 mM) group. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons, *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 10

Effect of butyrate on inflammatory response and cell neurotoxicity in BV2 cells induced by LPS. A Schematic illustration of experimental design. B–E The levels of cytokines (TNF-α, IL-6, IL-4, and IL-10) in the BV2 cells (n = 5). F–H Relative protein levels of HDAC3, p-IκB and p-P65 in the BV2 cells (n = 6). I Relative protein levels of cleaved caspase-3 in the HT22 cells (n = 6). TAK-242: TLR4 inhibitor, PDTC:NF-κB antagonists, AZD3965:MCT1 inhibitor, ITSA-1: HDAC3 agonist. The data represent the mean ± SEM, p < 0.05 was set as the threshold for significance by one-way ANOVA followed by post hoc comparisons using Tukey’s test for multiple groups’ comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the control group. # p < 0.05, ## p < 0.01, ### p < 0.001 compared to LPS group. + p < 0.05, +  + p < 0.01, +  +  + p < 0.001 compared to LPS + Mel group

Fig. 11

Schematic diagram of the protective effects of melatonin on cognitive impairment caused by sleep deprivation through the microbiota–gut–brain axis. Briefly, gut microbes and their metabolites mediate the ameliorative effect of melatonin on SD-induced cognitive impairment. A feasible mechanism is that Mel downregulates the Aeromonas population and production of the constituent LPS production and upregulates the Lachnospiraceae_NK4A136 population and the production of the butyrate metabolite by remodeling gut microbiota homeostasis. These events inhibit the TLR4/HDAC3/NF-κB signaling pathway, thereby preventing neuroinflammation and ultimately alleviating neuronal apoptosis and memory impairment in sleep-deprived mice. HDAC3: histone deacetylase3, LPS: lipopolysaccharide, Mel: melatonin, NF-κB: nuclear factor-κB, SD: sleep deprivation, TLR4: Toll-like receptor 4