Dysrhythmic saliva microbiota in mobile phone addicts with sleep disorders and restored by acupuncture

Abstract

Background: Mobile phone addiction (MPA) greatly affects the biological clock and sleep quality and is emerging as a behavioral disorder. The saliva microbiota has been linked to circadian rhythms, and our previous research revealed dysrhythmic saliva metabolites in MPA subjects with sleep disorders (MPASD). In addition, acupuncture had positive effects. However, the dysbiotic saliva microbiota in MPASD patients and the restorative effects of acupuncture are unclear.

Objectives: To probe the circadian dysrhythmic characteristics of the saliva microbiota and acupunctural restoration in MPASD patients.

Methods: MPASD patients and healthy volunteers were recruited by the Mobile Phone Addiction Tendency Scale (MPATS) and the Pittsburgh Sleep Quality Index (PSQI). Saliva samples were collected every 4 h for 72 h. After saliva sampling, six MPDSD subjects (group M) were acupuncturally treated (group T), and subsequent saliva sampling was conducted posttreatment. Finally, all the samples were subjected to 16S rRNA gene sequencing and bioinformatic analysis.

Results: Significantly increased MPATS and PSQI scores were observed in MPDSD patients (p< 0.01), but these scores decreased (p<0.001) after acupuncture intervention. Compared with those in healthy controls, the diversity and structure of the saliva microbiota in MPASD patients were markedly disrupted. Six genera with circadian rhythms were detected in all groups, including Sulfurovum, Peptostreptococcus, Porphyromonas and Prevotella. There were five genera with circadian rhythmicity in healthy people, of which the rhythmicities of the genera Rothia and Lautropia disappeared in MPASD patients but effectively resumed after acupuncture intervention.

Conclusions: This work revealed dysrhythmic salivary microbes in MPASD patients, and acupuncture, as a potential intervention, could be effective in mitigating this ever-rising behavioral epidemic.

Keywords: 16S rRNA gene sequencing; acupuncture; circadian rhythm; mobile phone addiction with sleep disorder (MPASD); saliva microbiota.

Figure 1

Flow chart of the experimental design.

Figure 2

The location of acupoints. (A) Neiguan (PC6). (B) Baihui (GV20). (C) Hegu (LI4). (D) Shenting (GV24). (E) Yintang (GV29). (F) Sanyinjiao (SP6). (G) Shenmen (HT7). (H) Anmian (EX-HN22).

Figure 3

Misaligned community of saliva microbes in MPASD patients restored by acupuncture intervention. The α diversity of the saliva microbiota was displayed by Chao1 (A), observed species (B), Shannon (C) and Simpson (D) indices (nonparametric Kruskal−Wallis test, p>0.05). (E) Principal component analysis (PCA) using unweighted UniFrac showed structural differences in the saliva microbiota (p<0.05). The circadian oscillations among the three groups were analyzed based on the α diversity coefficient and are presented as the Shannon (F), Chao1 (G) and ACE (H) indices. Shared and unique OTUs are shown in a Venn diagram (I). *p < 0.05.

Figure 4

Compositions of the saliva microbiota in groups N, M and T. The overall compositions of the saliva microbiota in groups N, M and T are represented as bar plots at the phylum (A) and genus (B) levels.

Figure 5

Saliva microbes with circadian rhythmicity. (A) The Venn diagram above shows the number of saliva microbes with circadian rhythmicity (upper), the intersection size (middle) and the various combinations (bottom) (p< 0.05). (B) The bubble map shows 19 unique genera with circadian rhythmicity in group M. The phase (C), amplitude (D) and period (E) of saliva microbes with circadian rhythmicity (p > 0.05) derived from groups M, T and N. ^ns p>0.05.

Figure 6

Visual curves of the circadian rhythms in saliva microbes obtained by cosine analysis. The genera Porphyromonas (A) and Prevotella (B) exhibited typical circadian rhythmicity in all groups. (C) The genus Haemophilus displayed circadian rhythmicity in healthy volunteers (group N) and was disrupted in MPASD subjects (group M) but restored after acupuncture intervention (group N). The cosine models were based on precise circadian phase data. To demonstrate the adequacy of these models in fitting the actual data, average data grouped into 60-circadian degree windows (approximately 4-hour resolution) were also plotted. The bottom X-axis indicates the circadian phase, with 0° representing the nocturnal 0 point of the fit. The top X-axes indicate the corresponding average clock time in these participants, and the Y-axis corresponds to the cosine calculated from the relative abundance of each group.