高脂饮食加重牙周炎对肠道菌群及糖代谢影响的实验研究

High-fat diet promotes the impact of periodontitis on gut microbiota and glucose metabolism

目的研究高脂饮食是否加剧牙周炎对肠道菌群及糖代谢的影响。方法将24只雄性SD大鼠通过随机数字表法随机分为4组(每组6只):对照组,给予常规饮食;牙周炎组,用5-0号丝线结扎大鼠双侧上颌第二磨牙诱导牙周炎;高脂饮食组,给予高脂饮食;高脂饮食+牙周炎组,给予高脂饮食,并在实验开始后8周诱导牙周炎。12周后检测空腹血糖及葡萄糖耐量。安乐处死大鼠,收集盲肠内容物,通过Illumina MiSeq平台对样本中16S rRNA基因进行测序,用RDP Classifier和SILVA(SSU123)16S rRNA数据库对测序结果进行分析。通过相关性分析研究高脂饮食和牙周炎诱导的肠道菌群变化与血糖水平的相关性。结果结扎诱导牙周炎4周后,牙周炎组大鼠和高脂饮食组大鼠的空腹血糖水平[分别为(4.93±0.28)、(5.25±0.24)mmol/L]均显著高于对照组大鼠的空腹血糖水平[(4.56±0.20)mmol/L](P<0.05),且牙周炎组大鼠和高脂饮食组大鼠均存在葡萄糖耐量受损。高脂饮食+牙周炎组的空腹血糖水平[(5.53±0.14)mmol/L]显著高于牙周炎组和高脂饮食组(P<0.05),其葡萄糖耐量曲线也高于牙周炎组。肠道菌群分析显示,牙周炎组大鼠肠道菌群中拟杆菌门与厚壁菌门的比值(0.37±0.23)显著低于对照组(0.68±0.05)(P<0.05),毛螺菌科NK4A136组的丰度[(14.03±6.38)%]显著低于对照组[(28.21±4.82)%](P<0.05),异杆菌属、瘤胃球菌科UCG_005、布劳特菌属的丰度[分别为(4.27±2.67)%、(3.70±0.90)%、(0.63±0.45)%]均显著高于对照组[分别为(0.60±0.72)%、(0.43±0.16)%、(0.13±0.13)%](P<0.05)。高脂饮食+牙周炎组大鼠肠道菌群中变形菌门的丰度[(3.06±0.90)%]显著高于牙周炎组[(1.40±0.98)%](P<0.05)。基于Bray-Curtis距离的β多样性分析显示,高脂饮食+牙周炎组大鼠的肠道菌群与牙周炎组、高脂饮食组均有明显的分群。相关性分析结果显示,肠道菌群中毛螺菌科NK4A136组的丰度与空腹血糖及负荷60和120 min的血糖水平呈显著负相关(r值分别为-0.56、-0.50、-0.42,P<0.05),异杆菌属、产粪甾醇真杆菌属、未培养的消化链球菌科、扭链瘤胃球菌属以及变形菌门中多个菌属的丰度与负荷120 min的血糖水平呈显著正相关(P<0.05)。结论牙周炎与肠道菌群紊乱和糖代谢异常密切相关,高脂饮食可使这种联系更为紧密。

Objective To study whether high-fat diet could aggravate the effect of periodontitis on gut microbiota and glucose metabolism.Methods Twenty-four male SD rats were randomly and equally divided into four groups based on table of random numbers(n=6 in each group):control group,in which rats were given normal chow diet;periodontitis group,in which periodontitis was induced by ligating bilateral maxillary second molars with 5-0 silk thread;high-fat diet group,in which rats were given high-fat diet;high-fat diet+periodontitis group,in which rats were given high-fat diet and periodontitis was induced at the end of the 8th week.Fasting blood glucose and glucose tolerance were measured at the end of the 12th week.Then the rats were euthanized and the cecum content was collected.The microbial 16S rRNA gene sequencing was performed on the Illumina MiSeq platform.The taxonomy of the sequences was analyzed through RDP Classifier(http://rdp.cme.msu.edu/)against the SILVA(SSU123)16S rRNA database.Pearson correlation analysis was performed to analyze the correlation between changes in gut microbiota and blood glucose.Results After 4 weeks of periodontitis induction,the fasting blood glucose levels of the periodontitis group and the high-fat diet group were(4.93±0.28)and(5.25±0.24)mmol/L,respectively,which were significantly higher than that of the control group[(4.56±0.20)mmol/L](P<0.05)with glucose intolerance.The fasting blood glucose level of high-fat diet+periodontitis group[(5.53±0.14)mmol/L]was significantly higher than that of periodontitis group and high-fat diet group,respectively(P<0.05),with the glucose tolerance curve higher than that of periodontitis group.The 16S rRNA gene analysis revealed that the Bacteroides/Firmicutes ratio in the periodontitis group is(0.37±0.23),which was significantly lower than that of the control group(0.68±0.05)(P<0.05).The relative abundance of Lachnospiraceae_NK4A136_group in the periodontitis group was(14.03±6.38)%,which was significantly lower than that of the control group[(28.21±4.82)%](P<0.05).The relative abundance of Allobaculum[(4.27±2.67)%],Ruminococcaceae_UCG_005[(3.70±0.90)%],Blautia[(0.63±0.45)%]in the periodontitis group were significantly higher than those of the control group[(0.60±0.72)%,(0.43±0.16)%,(0.13±0.13)%,respectively](P<0.05).Compared with periodontitis group,the relative abundance of Proteobacteria in high-fat diet+periodontitis group[(3.06±0.90)%]was significantly higher than that of the periodontitis group[(1.40±0.98)%](P<0.05).The principal coordinate analysis and similarity analysis based on the Bray-Curtis distance showed that samples of the high-fat diet+periodontitis group clustered separately from the periodontitis group and the high-fat diet group.The results of correlation analysis showed that the abundance of Lachnospiraceae_NK4A136_group was negatively correlated with fasting blood glucose and glucose levels after loading for 60 and 120 minutes(r=-0.56,-0.50,-0.42,respectively)(P<0.05).The abundance of Allobaculum,[Eubacterium]_coprostanoligenes_group,Peptococcaceae_uncultured,[Ruminococcus]_torques_group,and several genera belonging to the Proteobacteria were positively correlated with glucose levels after loading for 120 minutes(P<0.05).Conclusions Periodontitis might be closely related to impaired gut microbiota and glucose metabolism,and the effect could be aggravated by high-fat diet.