感冒风热证与风热夹湿证体质和唾液菌群差异研究

Differences in Constitution and Salivary Microbiota between Windheat Syndrome and Wind-heat Dampness Syndrome of Cold

目的通过对感冒风热证(WH)与风热夹湿证(WW)患者的症状表现、体质状态、免疫状态和唾液菌群进行研究分析,探讨两种证候的物质基础差异。方法采用临床病例收集的方法,记录患者的一般信息、临床资料,运用统计学方法,研究两种证型的症状分布特点、体质状态差异。采集感冒患者的血液样本并检测血液指标,比较两个证型的免疫差异。收集患者的唾液,利用16S rRNA分析唾液菌群的差异。采用PICRUSt法进行分析,对照京都基因与基因组百科全书(KEGG)数据库进行比较,预测唾液菌群代谢能力。结果15例WH患者和15例WW患者被纳入研究,两组患者的体质和症状评分存在差异(P<0.05)。免疫指标对比,γ干扰素(IFN-γ)、IFN-γ/白细胞介素4(IL-4)风热夹湿证组明显高于风热证组(P<0.05)。Alpha多样性中,observed species和Chao1的WH组均高于WW组(P<0.05)。分类树热图显示在门水平上拟杆菌门(Bacteroidetes)和厚壁菌门(Firmicutes),在目水平上梭状芽孢杆菌目(Clostridiales),在科水平上的卟啉单胞菌科(Porphyromonadaceae),在属水平上的普氏菌属(Prevotella)、Catonella、Eggerthia和密螺旋体属(Treponema)WH组的相对丰度高于WW组(P<0.05)。物种丰度表计算结果显示,在门水平上梭杆菌门(Fusobacteria)、螺旋体门(spirrochaetes)、互养菌门(synnergistetes)和绿弯菌门(Chloroflexi),在纲水平上梭杆菌纲(Fusobacteriia)、螺旋体纲(Spirochaetia)、拟杆菌纲(Bacteroidetes)和互养菌纲(Synergistia),在目水平上梭杆菌目(Fusobacteriales)和螺旋体目(Spirochaetales),在属水平上普氏菌属(Prevotella)WH组的相对丰度高于WW组(P<0.05)。在top50功能中,转运蛋白、其他蛋白、肽聚糖生物合成、泛酸和辅酶a生物合成以及糖酵解/糖异生等功能WH组均低于WW组(P<0.05)。其他离子偶联转运体、能量代谢、膜和细胞内结构分子、卟啉和叶绿素代谢以及孔道离子通道WH组均高于WW组(P<0.05)。结论研究结果表明,风热证与风热夹湿证患者的症状、体质状态具有差异性,风热夹湿证患者体内Th1细胞介导的促炎反应较风热证组强。感冒风热证与风热夹湿证的唾液菌群存在差异,中医证型可能与机体免疫状态具有一定的相关性。

Objective To explore the material basis differences between the wind-heat syndrome(WH)and wind-heat dampness syndrome(WW)of patients with cold through comparing the symptoms,constitution states,immune states and salivary microflora.Methods The general information and clinical data of the patients were recorded by the method of clinical case collection,and the differences in symptom distribution and constitution status between the two syndrome types were studied by statistical methods.Blood samples were collected from patients with cold and blood indexes were detected to compare the immune differences between the two syndromes.Saliva was collected from the patients and 16S rRNA was used to analyze the differences in salivary microbiota.PICRUSt method was used for analysis and KEGG database was used for comparison to predict the metabolic capacity of salivary microbiota.Results 15 WH patients and 15 WW patients were enrolled in the study.There were differences in physique and symptom scores between the two groups(P<0.05).The immunological indexes of IFN-γand IFN-γ/IL-4 were significantly higher in WW group than in WH group(P<0.05).In Alpha diversity,WH group of observed species and Chao1 were higher than WW group(P<0.05).The classification tree heat map showed that Bacteroidetes and Firmicutes at phylum level;Clostridiales at order level;Porphyromonadaceae at family level;Prevotella,Catonella,Eggerthia and Treponema at genus level,the abundance of WH group was higher than WW group(P<0.05).The results of species abundance table calculation showed that Fusobacteria,Spirochaetes,Synergistetes and Chloroflexi at phylum level;Fusobacteria,Spirochaetia, Bacteroidetes and Synergistia at class level. Fusobacteriales and Spirochaetales at order level. Prevotella at genus level,the relative abundance in WH group was higher than that in WW group(P <0.05). Transporters,others,peptidoglycan biosynthesis,pantothenate and CoA biosynthesis and glycolysis/ gluconeogenesisare among the top50 functions in WH group were lower than that in WW group(P <0.05). Other ion-coupled transporters,energy metabolism,membrane and intracellular structural molecules, porphyrin and chlorophyll metabolism and pores ion channels in WH group were higher than that in WW group(P <0.05). Conclusion Our results showed that the symptoms and constitution status of patients with wind-heat syndrome and wind-heat dampness syndrome were different,and the Th1 cell-mediated pro- inflammatory response in patients with wind-heat syndrome was stronger than that in wind-heat dampness syndrome group. There are differences in salivary microbiota between wind-heat syndrome and wind-heat dampness syndrome. TCM syndromes may be correlated with immune status and salivary microbiota.