PM2.5 induced neurotoxicity through unbalancing vitamin B12 metabolism by gut microbiota disturbance

Abstract

Fine particulate matter (PM_2.5) in the atmosphere is easily accompanied by toxic and harmful substances, causing serious harm to human health, including cognitive impairment. Vitamin B12 (VitB12) is an essential micronutrient that is synthesized by bacteria and contributes to neurotransmitter synthesis as a nutrition and signaling molecule. However, the relationship between VitB12 attenuation of cognitive impairment and intestinal microbiota regulation in PM_2.5 exposure has not been elucidated. In this study, we demonstrated that PM_2.5 caused behavioral defects and neuronal damage in Caenorhabditis elegans (C. elegans), along with significant gene expression changes in neurotransmitter receptors and a decrease in VitB12 content, causing behavioral defects and neuronal damage in C. elegans. Methylcobalamin (MeCbl), a VitB12 analog, alleviated PM_2.5-induced neurotoxicity in C. elegans. Moreover, using in vivo and in vitro models, we discovered that long-term exposure to PM_2.5 led to changes in the structure of the gut microbiota, resulting in an imbalance of the VitB12-associated metabolic pathway followed by cognitive impairment. MeCbl supplementation could increase the diversity of the bacteria, reduce harmful substance contents, and restore the concentration of short-chain fatty acids (SCFAs) and neurotransmitters to the level of the control group to some degree. Here, a new target to mitigate the harm caused by PM_2.5 was discovered, supplying MeCbl for relieving intestinal and intracellular neurotransmitter disorders. Our results also provide a reference for the use of VitB12 to target the adjustment of the human intestinal microbiota to improve metabolic disorders in people exposed to PM_2.5.

Keywords: Vitamin B12; atmospheric particulate matter; gut microbiome; in vitro colonic simulation system; methylcobalamin; neurotransmitters.

Figure 1.

Effects of MeCbl on PM_2.5-induced toxicity in C. elegans. (a-c) the impact of MeCbl intervention on body bending times (1 min), head swing times (20 s) and pharyngeal pumping rate (20 s) of worms. *p < .05; **p < .01; ns: not significant by Student’s t-test. (d) lifespan assay on worms with different treatments. (e) chemotaxis-mediated associative learning model; (f) chemotaxis learning behavior after 72 h PM_2.5 exposure with or without MeCbl; (g) the schematic diagram for analyzing chemotaxis of C. elegans. (h) chemotaxis behavior of C. elegans after 72 h PM_2.5 exposure with or without MeCbl; indole and decanal (dissolved in ethanol) were used for studying the sensitivity of nematodes to chemicals; (i) the schematic diagram for analyzing foraging behavior of C. elegans; (j) foraging behavior after 72 h PM_2.5 exposure with or without MeCbl. Values indicated by the bars with different letters are significantly different (p < .05, one-way ANOVA). CI, chemotaxis index.

Figure 2.

Effects of MeCbl on GABAergic, cholinergic and glutamatergic neurons of EG1285, LX929and DA1240 after a 72 h PM_2.5-exposure period. (a) Fluorescent photographs show the GABAergic neuron in worms when exposed to PM_2.5 with or without MeCbl treatment. (b) Neuronal injury in EG1285 nematodes after PM_2.5 and MeCbl treatment. (c) Fluorescent photographs show the cholinergic neuron in worms when exposed to PM_2.5 with or without MeCbl treatment. (d) Neuronal injury in LX929nematodes after PM_2.5 and MeCbl treatment. Degree of neuronal injury was classified into three categories, namely normal (good neuronal integrity), intermediate (degradation of a few neurons, less than 5), abnormal (more than 5 neurons degradation, discontinuous or misplacement). (e) Fluorescent photographs show the glutamatergic neuron in worms when exposed to PM_2.5 with or without MeCbl treatment. (f) Relative fluorescence intensity of eat-4:GFP in C. elegans with indicated treatments. Values indicated by the bars with different letters are significantly different (p < .05, one-way ANOVA).

Figure 3.

Effects of MeCbl on the mRNA levels of genes related with cobalamin metabolism and neurotransmitters in C. elegans exposed to PM_2.5. (a) Levels of Met/SAM cycle genes affected by PM_2.5 and MeCbl. (b) Cartoon of VitB12-related metabolic pathways in C. elegans. (c) Effect of MeCbl on neurotransmission-related gene expression in nematodes exposed to PM_2.5. Relative gene expressions were normalized to act-1 gene. (d) Fluorescent photographs and relative fluorescence intensity of Pacdh1:GFP transgenic animals (VL749 strain) when exposed to PM_2.5 with or without MeCbl treatment. The acyl-CoA dehydrogenase acdh-1 is a well-established sensor gene for B12 levels, and a lower B12 level triggers higher expression of acdh-1. Values indicated by the bars with different letters are significantly different (p < .05, one-way ANOVA).

Figure 4.

Effects of MeCbl on constitutes of intestinal bacteria and metabolite in mice exposed to PM_2.5. (a) α-diversity-Shannon index of fecal microbiota; (b) Heatmap of relative abundance of bacterial genus in the intestinal tract of mice; (c) Principal component analysis of intestinal metabolites in mice (the difference in PCA1 and PCA2 was 53.43% and 45.75%); (d) Intestinal short-chain fatty acid concentration in mice; (e) metabolite heatmap of intestinal microbiome in mice; (f) Correlation analysis of metabolites and intestinal microflora in mice.

Figure 5.

Effect of MeCbl on changes in the composition of bacteria and metabolites in gut lumen of the in vitro colon simulation system exposed to PM_2.5. (a) histogram of changes in SCFAs in the gut lumen of the colon; (b) cartoon of relationship between butyrate production and gut flora changes impacted by PM_2.5 and MeCbl; (c) heatmap of relative abundance of bacteria genus; (d) heatmap of secondary metabolic intensity of bacteria; (e) correlation analysis of metabolites and intestinal microflora; (f) canonical correlation analysis (CCA) between species and neurotransmitters. We firstly carry out detrended correspondence analysis (DCA) analysis on data and the DCA1 of relevant data in this study is 3.7928 that is greater than 3, so we choose a CCA model.

Figure 6.

Toxin, enzyme activity and VitB12 contents of in in vitro colon simulation system at different periods of fermentation. (a) LPS contents; (b) Shiga toxin contents; (c) protease activity; (d) amylase activity; (e-h) line chart of four types of VitB₁₂ (cyanocobalamin, methylcobalamin, Adenosylcobalamin and hydroxocobalamin) content changes.

Figure 7.

Changes of cobalamin metabolism and transport in vitro colon simulation system (lumen) by KEGG pathways analysis (day 4). (a) genes of cobalamin metabolic enzymes up-regulated by PM_2.5 exposure; (b) genes of cobalamin metabolic enzymes down-regulated by PM_2.5 exposure; MeCbl solution was added to the fermenter at the final concentration of 0.0125 mg/mL (MeCbl-L) and 1.25 mg/mL (MeCbl-H); (c) key pathways associated with cobalamin metabolism. Up-regulated genes were marked in red and down-regulated genes were marked in blue after PM_2.5 treatment, while all these marked genes level were significantly reversed by MeCbl supplementation; (d) flowchart of key pathway of bacterial B12 synthesis. Bacteria listed here was significant changed in PM_2.5 and MeCbl-treated groups; (e) diagram of the VitB12 transport machinery in E. coli. (f) relative transcriptional levels of BtuB, TonB, ExbD and ExbB that involved in VitB12 transport in fermentation at day 4. Values indicated by the bars with different letters are significantly different (p < .05, one-way ANOVA).