Proteomic and microbiota analyses of the oral cavity during psychological stress

Abstract

Psychological stress is associated with various oral diseases such as aphthous stomatitis, oral lichen planus, taste disturbances and glossodynia. However, the underlying mechanism is still unknown. The aim of this study was to determine the effect of psychological stress on salivary proteins and the oral microbiota in a rat model of chronic restraint stress. Six-week-old Sprague Dawley rats were subjected to restraint stress for four hours daily for 1 month. The behavior, weights of the adrenal glands, and serum corticosterone levels were evaluated as stress markers. Proteomic analysis of the saliva was performed using two-dimensional gel electrophoresis followed by mass spectrometry and Western blotting. Analysis of the oral microbiota was performed via 16S rRNA next-generation sequencing. The low mean body weights, lower number of entries and time spent in the open arm of elevated plus maze, high adrenal gland/body weight ratios, and high serum corticosterone levels confirmed the high levels of stress in the stress group of rats compared to the controls. Thirty-three protein spots were found to be significantly altered between the two groups. After silver staining, seven visible spots were subjected for mass spectrometry, and the expression levels of the two most significantly altered proteins, BPI fold containing family A member 2 and von Ebner’s gland protein, were confirmed by Western blotting. 16S rRNA sequencing analysis revealed a significant reduction in alpha diversity in the stress group compared to the controls. The abundances of oral bacteria, such as Facklamia and Corynebacterium, were significantly altered between the two groups. Additionally, analysis with PICRUSt2 software predicted 37 different functional pathways to be altered between the groups. In conclusion, the present study identified altered salivary proteins and oral microbiota due to psychological stress. These findings might aid in understanding the pathogenesis of stress-related oral diseases.

Effect of psychological stress on the oral-gut microbiota and the potential oral-gut-brain axis

“…No significant differences in the beta diversities of the oral microbiome were observed between anxiety and depression patients with high and low symptoms [66] . A separate, but statistically non-significant, clustering was observed between the stress and control groups in an in vivo experiment using a rat model of chronic restraint stress [61] . However, a clinical study performing a constrained analysis of the oral microbiome reported significant clustering between the healthy and depressed cohorts [62] .…”

“…Thus, using an animal model might overcome the differences in local and systemic factors as seen in humans and provide reliable data on the alterations in the oral microbiome due to psychological stress. In our recent study comprising a rat model of chronic restraint stress, 16 S rRNA sequencing of oral microbiota revealed that the alpha diversity as measured by observed operational taxonomic unit and faith phylogenetic diversity was significantly lower in the stress group when compared to that in the control group [61] . Consistent with our findings, a previous experiment on wild red squirrels showed that the alpha diversity of the oral microbiome was lower in those with elevated fecal glucocorticoid metabolites, a measure of the hypothalamic-pituitary-adrenal axis [60] .…”

“…A significant reduction in the aggregation of S. gordonii was observed in unstimulated whole saliva collected 10 min before an academic examination compared to that collected 2 and 6 weeks after the examinations [74] . Facklamia and Corynebacterium were reported to be significantly increased and decreased, respectively, in the oral cavities of rats with chronic resistant stress when compared to those in the control group [61] .…”

“…Various preclinical ( Table 1 ) [60] , [61] and clinical studies ( Table 2 ) [62] , [63] , [64] , [65] , [66] , [67] , [68] , [69] , [70] , [71] , [72] have demonstrated the effect of psychological stress on the oral microbiome via alterations in the diversity and taxonomic abundance of the bacteria. The alpha diversity measures the richness or evenness of the species within a sample, which is commonly determined by 16 S rRNA sequencing.…”

“…It remains unclear as to how the bacteria migrated to hippocampus; nonetheless, it is thought that the oral bacteria may have used the trigeminal route to reach the brain. Corynebacterium , a bacterium that produces serotonin in the gut, was found to be decreased in the oral cavities of rats exposed to chronic restraint stress [61] . The reduction of oral Corynebacterium that possibly produces serotonin in the chronic stress is of interest in oral-brain axis.…”