Fluoride Action Network



  • Memory function was reduced in the fluoride-exposed mice.
  • Gut microbiota structure in fluoride-exposed mice was significantly altered.
  • Lactobacillus was the mainly targeting bacteria in fluoride-treated mice.
  • Inoculation with Lactobacillus johnsonii reconstructed gut microbiota in fluoride-exposed mice.
  • Inoculation with Lactobacillus johnsonii rescued memory dysfunction in fluoride-expose mice.

Fluoride which is widespread in our environment and food due to its geological origin and industrial pollution has been identified as a developmental neurotoxicant. Gut-brain axis provides new insight into brain-derived injury. We previously found the psychoactive effects of a probiotic strain, Lactobacillus johnsonii BS15 against fluoride-induced memory dysfunction in mice by modulating the gut-brain axis. In this study, we aimed to detect the link between the reconstruction of gut microbiota and gut-brain axis through which probiotic alleviate fluoride-induced memory impairment. We also added an hour of water avoidance stress (WAS) before behavioral tests and sampling, aiming to demonstrate the preventive effects of the probiotic on fluoride-induced memory impairment after psychological stress. Mice were given fluoridated drinking water (sodium fluoride 100 ppm, corresponding to 37.8 ± 2.4 ppm F¯) for 70 days and administered with PBS or a probiotic strain, Lactobacillus johnsonii BS15 for 28 days prior to and throughout a 70 day exposure to sodium fluoride. Results showed that fluoride increases the hyperactivity of hypothalamic-pituitary-adrenal (HPA) axis and reduces the exploration ratio in novel object recognition (NOR) test and the spontaneous exploration during the T-maze test in mice following WAS, which were significantly improved by the probiotic. 16S rRNA sequencing showed a significant separation in ileal microbiota between the fluoride-treated mice and control mice. Lactobacillus was the main targeting bacteria and significantly reduced in fluoride-treated mice. BS15 reconstructed the fluoride-post microbiota and increased the relative abundance of Lactobacillus. D-lactate content and diamine oxidase (DAO) activity, two biomarkers of gut permeability were reduced in the serum of probiotic-inoculated mice. ZO-1, an intestinal tight junction protein was reduced by fluoride in mRNA, and its protein levels were increased by the probiotic treatment. Moreover, the hippocampus which is essential to learning and memory, down-regulated mRNA level of both the myelin-associated glycoprotein (MAG), and protein levels of brain-derived neurotrophic factor (BDNF), including the improvement of cAMP response element-binding protein (CREB) by BS15 in fluoride-exposed mice after WAS. Via spearman correlation analysis, Lactobacillus displayed significantly positive associations with the behavioral tests, levels of nerve development related factors, and intestinal tight junction proteins ZO-1, and negative association with TNF-? of the hippocampus, highlighting regulatory effects of gut bacteria on memory potential and gut barrier. These results suggested the psychoactive effects of BS15 on fluoride-induced memory dysfunction after psychological stress. In addition, there may be some correlations between fluoride-induced memory dysfunction and reconstruction of gut microbiota.

Availability of data and materials

16S rRNA sequencing reads have uploaded to NCBI. The accession code of 16S rRNA sequencing reads in the National Center for Biotechnology Information (NCBI) BioProject database: PRJNA660154.


Lactobacillus johnsonii BS15
Memory dysfunction
Gut–brain axis
Gut microbiota


1. Introduction

Fluoride is a widespread environmental pollution, and groundwater is the major source of exposure in which the fluoride concentration can be as high as 35 ppm (Petrone et al., 2013). Fluorosis induced by geological origin is a serious public health concern in 28 nations particularly in India and China (Rafique et al., 2015). In India, 230 districts of 20 states are at risk of a high level of fluoride in drinking water (Srivastava and Flora, 2020). In China, almost all the provinces have reported fluorosis except for Shanghai and Hainan (Sun, 2010, Sun et al., 2009). Moreover, social modernization results in fluoride pollution because of industrial production, the mechanical processing of food, and the use of fluorine-containing crop protection. Based on previous reports, fluoride concentration in canned meat and brick tea is under 1 to more than 8.6 mg/kg (Fein and Cerklewski, 2001) and 600–2800 mg/kg (Fung et al., 1999), respectively. Fluoride accumulation in our body can damage both bone (Petrone et al., 2013) and non-bone tissues (Yan et al., 2019, Qian et al., 2013), such as the liver, kidney, spleen, and brain. The neurotoxic effects of fluorine must not be ignored because such effects can affect brain health in rodents at levels below those that induce dental lesions (Grandjean, 2019). To date, fluoride has been identified as a developmental neurotoxicant (Grandjean and Landrigan, 2014).

Recently, there is increasing evidence on fluoride-induced brain damage by focusing on learning and memory dysfunction. An epidemiological study from Hulunbuir, Inner Mongolia of China based on 331 children aged 7–14 from four schools with the same teaching quality demonstrated that fluoride exposure even in low levels had negative effects on children’s memory (Ding et al., 2011). Similar studies were also found in other countries (Green et al., 2020, Bashash et al., 2017). Moreover, Liu et al. (2010) found that a rat exposed to fluoride (50 ppm NaF) for 6 months showed prolonged escape latency in the Morris water maze test. Other rodent experiments also demonstrated that fluoride exposure could cause microtubule lesions; thickened postsynaptic density; pathologic, indistinct, and short synaptic cleft; and myelin damage (Niu et al., 2018). Furthermore, brain-derived neurotrophic factor (BDNF) and cAMP/Ca2+-responsive element-binding protein (CREB), which have been identified to be involved in hippocampal plasticity and hippocampus-dependent memory based on considerable evidence, were decreased in mice exposed to fluoride (100 ppm NaF) for 60 days (Niu et al., 2018).

Moreover, fluoride-exposed mice were not only confronted by memory impairment but also accompanied by intestinal inflammation and increased gut permeability. It is well accepted that intestinal microbiota highly shapes the intestinal microenvironment including intestinal barrier function and intestinal inflammation (Xin et al., 2020). Interestingly, increasing evidence has demonstrated that the gut microbiota is associated with mood and memory disturbances, and improving the gut microbiota is a potential method to treat such diseases. For example, patients with colitis characterized by disordered gut microbiota have a high risk for anxiety (21%) and depression (15%) (Neuendorf et al., 2016). Different colitis models represent the human behavioral phenotype. Zhao et al. (2020) found that the depression and anxiety-like behavior in dextran sulfate sodium-induced colitis model could be improved by lycopene through increasing the relative abundance of Bifidobacterium and Lactobacillus. Similarly, Jang et al. (2018) inoculated Lactobacillus johnsonii to 2,4,6-trinitrobenzenesulfonic acid-induced colitis model, which improved memory impairment by restoring the disturbed gut microbiota composition. Neuroactive metabolites and gut integrity are the main mechanisms underlying the communication between the gut and brain. Recently, Mao et al. (2020) observed increased levels of lactate in the fecal and brains of mice inoculated with Lactobacillus, and consequently, the mice had an improved memory. High levels of GABA are linked to novel object recognition and improved working memory and are consumed and produced by the gut microbiota, which influences circulating GABA levels (Strandwitz, 2018). Damage to gut integrity can cause bacteria and harmful metabolites to enter the brain. Recently, Emery et al. (2017) found evidence for microbiological incursion into the brain. Zhan et al. (2016) found increased levels of Escherichia coli K99 and lipopolysaccharide (LPS) in Alzheimer’s disease (AD) brain and suggested that Gram-negative bacteria-derived LPS induced AD neuropathology in an ischemia–hypoxia rat model. Collectively, these studies indicate a link between the gut microbiota and memory potential.

Luo et al. (2016) found that Lactobacillus spp. remarkably decreased and E. coli and Enterococcus spp. increased in fluoride-treated broiler. Thus, studies based on gut-brain axis hypothesis may be effective in preventing the fluoride-associated memory impairment through the modulation of gut environment by probiotics. Lactobacillus. johnsonii BS15 (CCTTCC M2013663) was isolated from homemade yogurt collected from Hongyuan Prairie, Aba Autonomous Prefecture, China. L. johnsonii BS15 showed a steady effect on adjusting the gut environment and lowering the intestinal permeability of mice with high-fat diet, thereby preventing non-alcoholic fatty liver disease (Xin et al., 2014). L. johnsonii BS15 could also be considered as a potential “psychobiotic” as it was found to prevent psychological stress-induced memory dysfunction in mice by modulating the gut environment (Wang et al., 2020). Therefore, we selected L. johnsonii BS15 to regulate the gut environment, aiming to demonstrate the relationship between the intestinal environment and the memory impairment in fluoride-exposed mice. The psychoactive effect of L. johnsonii BS15 against fluoride-exposed memory dysfunction through gut-brain axis was revealed in our previous study (Xin et al., 2020). However, to further understand the mechanism underlying the psychoactive effect, it is still important to establish the microbiome-gut-brain axis by detecting the reconstruction of gut microbiota. Though the underlying mechanism remains elusive, a close relationship between psychological stress and intestinal inflammation has been widely accepted (Wu et al., 2014). Moreover, according to our previous study, memory dysfunction was found in mice subjected to 7-day water-avoidance stress (WAS), and the gut-brain axis was also significantly influenced (Wang et al., 2020). As part of our project to demonstrate the mechanism of fluoride-induced memory dysfunction and the effects of modulating gut-brain axis, we were also interested in determining whether or not the probiotic could alleviate fluoride-induced memory impairment after psychological stress.

Therefore, we used 16S rRNA gene sequencing to detect the feature of the gut microbiota in fluoride-treated mice and BS15-treated mice in the present study. In addition, we assessed the difference in memory ability between untreated and treated individuals by T-maze test and novel object recognition (NOR) test after psychological stress. We selected 1 h of WAS as the psychological stress since a single 1 h of WAS did not result in a measurable change in memory ability but might aggravate the memory impairment when other stressor was given (Gareau et al., 2011). In addition to evaluating the intestinal integrity and permeability, hippocampal inflammation and memory-associated protein were detected in this study given the importance of the hippocampus on memory function. The present study aimed to provide evidence to answer the following two research questions: 1) Was the gut microbiota reconstructed by L. johnsonii BS15 when using the probiotic to modulate the gut-brain axis and alleviate fluoride-induced memory impairment? 2) Did the preventive effects of L. johnsonii BS15 still exist on fluoride-induced memory impairment after psychological stress?