Differential Analysis of Oral Microbiota in db/db Mouse Model of Type 2 Diabetes Utilizing 16S rRNA Sequencing

doi:10.12300/j.issn.1674-5817.2024.119

Abstract

Abstract:

Objective To investigate the changes in oral microbiota of db/db mice and provide an experimental basis for exploring the relationship between type 2 diabetes mellitus and oral microecology. Methods Eight 10-week-old male db/db mice were designated as the diabetes experimental group (db/db group), while eight 10-week-old male db/m mice were assigned as the normal control group (db/m group). After a 5-day adaptive feeding period, tail venous blood samples were collected on the 6th and 37th days, and fasting blood glucose (FBG) levels and oral glucose tolerance test (OGTT) were performed for both groups to verify the reliability of the diabetes model. On the 15th day of feeding with the same diet, oral microbiota samples were collected from the buccal mucosa, dorsal and ventral tongue surfaces, oral floor mucosa, hard palate mucosa, and the gingival areas of both the upper and lower jaws of the two groups. Genomic DNA from the oral microbiota was extracted, and the V3-V4 regions of the 16S ribosomal RNA (16S rRNA) gene were amplified using a GeneAmp 9700 thermocycler. The composition of the oral microbiota was evaluated through double-labelled amplification and sequencing on the Illumina MiSeq platform, followed by bioinformatics analysis using QIIME software(version 1.6.0). Results The FBG levels and OGTT results on the 6th and 37th days after the start of the experiment indicated that db/db mice exhibited more pronounced symptoms of type 2 diabetes compared to db/m mice. Alpha diversity (α diversity) analysis showed no significant difference in the diversity of oral microbiota between the two groups (P>0.05); however, there was a significant difference in richness (P<0.05). Principal coordinate analysis(PCoA) revealed differences in the oral microbiota composition between the db/db group and db/m group (P<0.05). Species composition analysis and LEfSe analysis demonstrated that the relative abundance of oral microbiota in db/db group mice, predominantly composed of p_Proteobacteria, increased significantly at the phylum level (P<0.05). At the genus level, the relative abundances of g_Proteus and g_Enterococcus showed a significant increase (P<0.001). Conclusion The composition and diversity of oral microbiota in db/db mice with type 2 diabetes mellitus significantly differed from those without the disease.

Key words: Type 2 diabetes, db/db mouse, Oral microbiota, 16S rRNA sequencing analysis

CLC Number:

Q95-33

PAN Qianjia,GE Junyi,HU Nan,et al. Differential Analysis of Oral Microbiota in db/db Mouse Model of Type 2 Diabetes Utilizing 16S rRNA Sequencing[J]. Laboratory Animal and Comparative Medicine, 2025, 45(2): 147-157. DOI: 10.12300/j.issn.1674-5817.2024.119.

Figures/Tables 5

Figure 1 Comparison of blood glucose indices in db/db mice and db/m miceNote：A， The concentration of fasting blood glucose （FBG） levels after a 12-hour fasting period； B， Analysis of oral glucose tolerance test （OGTT） from 0-2 hours； C， Area under the curve （AUC） of the OGTT. Db/db mice served as the diabetic group， while db/m mice were the normal controls， n=8 per group. *P＜0.05， **P＜0.01， ***P＜0.001.

Figure 2 PCoA and LEfSe analysis of oral microbiota composition in db/db mice and db/m miceNote：A， Principal coordinate analysis （PCoA） between db/db mice and db/m mice； B， LEfSe-derived cladogram showing taxonomic differences from phylum to genus level （p for phylum； c for class； o for order； f for family； g for genus）. Db/db mice served as the diabetic model， while db/m mice were the normal controls， n=8 per group.

Table 1 Alpha (α) diversity analysis of oral microbiota in db/db mice and db/m mice

指标 Index	db/m	db/db	P value
观察物种指数 Sobs	10.25±8.43	77.25±29.33	0.049^*
香农指数^# Shannon^#	2.40 (2.25~2.48)	2.32 (2.23~2.51)	0.529
辛普森指数 Simpson	0.18±0.04^*	0.16±0.04	0.499
基于丰度的覆盖估计值 ACE	105.56±8.34	79.49±30.06	0.045^*
Chao1丰富度估计量 Chao1	105.67±9.78	79.25±30.42	0.046^*
测序覆盖度 Coverage	0.999 9	1.000 0	0.458

Figure 3 Comparison of oral microbiota between db/db mice and db/m mice at the phylum levelNote：A-B， Phylum-level community composition of oral microbiota in db/db and db/m groups； C， Relative abundance of p_Proteobacteria. db/db mice served as the diabetic model， while db/m mice were the normal controls， n=8 per group. *P＜0.05.

Figure 4 Differences in oral microbiota composition between db/db mice and db/m mice at the genus levelNote： A， Heatmap showing the relative abundance of the top 50 dominant species in the saliva microbiota （db/db vs. db/m）； B-M， Relative abundance of the following genera： g_Staphylococcus， g_Muribacter， g_Streptococcus， g_Aerococcus， g_Corynebacterium， g_Jeotgalicoccus， g_Enterococcus， g_Proteus， g_Parabacteroides， g_Ruminococcaceae， g_Enterorhabdus， and g_Fictibacillus. Normally distributed data are presented as bar plots， and non-normally distributed data as box plots. Db/db mice served as the diabetic model， while db/m mice were the normal controls， n=8 per group. *P < 0.05， **P < 0.01， ***P < 0.001.

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