| Objective:To explore the pathological features of oral immunization and flora of patients with recurrent aphthous ulcer(RAU)of latent fire in the spleen and stomach,and provide a reference for exploring microscopic indicators of TCM syndromes from the perspective of micro dysbiosis;To clarify the role and mechanism of Xie-huang-San(the representative treatment of spleen and stomach fire)for regulating the oral flora of RAU patients and discover a new pharmacological function of classic prescriptions for regulating the flora;to establish a "syndrome-flora study" research platform and provide the new scientific evidence for the basic research and clinical application of relevant disease syndromes.Methods:Part 1: The clinical efficacy observation of RAU patients of latent fire in the spleen and stomach with fortifyingspleen and purging fire method.We included 62 patients who met the diagnostic criteria of RAU patients of latent fire in the spleen and stomach.All Patients were treated with Xie-huang-San for 5 days.The changes in scores of principal syndromes,scores of secondary syndromes,total scores,and long-term morbidity before and after treatment were compared to evaluate the clinical efficacy of Xie-huang-San for the treatment of RAU patients of latent fire in the spleen and stomach.Part 2: Experimental study on regulating immune dysregulation of RAU patients with latent fire in the spleen and stomach in the fortifyingspleen and purging fire method.Based on the the results of the first part,the 30 patients with curative effect were selected.Saliva of the 30 patients was collected before and after treatment,and saliva of 30 normal individuals were collected.A total 90 saliva samples were included in the study.After the pretreatment of the sample,the contents of sIgA,Lys,hBD-2,and TNF-α in all samples were detected by enzyme-linked immunosorbent assay(ELISA).The differences in immune function between RAU patients with latentfire in spleen and stomach and normal individuals were compared,and the regulating action on the immune dysregulation of the fortifying spleen and purging fire method in the patients was compared.Part 3: Study on role of the fortifyingspleen and purging fire method in regulating the oral microflora imbalance in RAU patients with latent fire in the spleen and stomach.By 16 S rRNA(amplicon sequencing)sequencing anaLysis,the differences in species relative abundance,the taxonomic trees of species,sample complexities,and diversities of different groups of oral microflora(phylum,class,order,family,genus,species)were observed.By Metagenome sequencing,the differences in the expression of important functional genes in the oral flora of each group were observed,and species categories,abundancewere analyzed,and related functions were interpreted.Results:(1)Compared with the numbers of ulcers,ulcer area,peripheral tissue congestion,ulcer pain and other local symptoms(principal syndromes)of RAU patients with latent fire in spleen and stomachbefore treatment,there were significant differences in that of patientstreated with Xie-huang-san(P<0.05).There were also found significant differences in the systemic symptoms(secondary syndromes)of heat in the mouth,halitosis,dry mouth,thirsty,yellow urine,and dry stool(P<0.05).The total effective rate of symptom improvement was 92.3% after treatment.The recurrence rate in 3 months was 71.43% after treatment compared with 96.64% before treatment.After treatment,the recurrence rate was significantly lower than rate before treatment,indicating that long-term recurrence time(recurrence rate in 3 months)was prolonged.No adverse reactions or side effects were discovered during the treatment.(2)The contents of sIgA,Lys,and hBD-2 in the saliva of RAU patients with latent fire in the spleen and stomach were significantly lower than those in the normal group.The difference was of great significance(P<0.01).There are partial and systemic immune dysfunction in RAU patients with latent fire in the spleen and stomach.After treatment of Xie-huang-San,the contents were significantly enhanced,which was close to normal range.The content of TNF-α in the saliva of the patientgroup was higher than that of the normal group(P<0.01),it was decreased after treatment too,but the difference was not significant(P>0.05).It suggested that the immunologic derangement in RAU patients was not completely corrected in the intermission of ulcers and there was also a persistent cellular immune dysfunction.(3)AnaLysis of relative abundance and cluster thermograms of oral flora was conducted.The change in species richness of oral microflora in RAU patients with latent fire in the spleen and stomach was obvious at portal level: Firmicutes,Proteobacteria,Bacteroidetes,Fusobacteria and spirochete.Changes in Proteobacteria and spirochete were higher than those in the normal group.The changes in Firmicutes,Bacteroidetes and Fusobacteria were lower than those in the normal group.At the genus level,the changes in Streptococcus,Prevotella,Bacaeroides,Fusobacterium,and Veillonia were all decreased compared with the normal group,while the changes of Haemophilus and Porphyromonas were higher than those in the normal group.There was no significant change in Neisseria,Rosinella,etc.The beneficial bacteria in the oral microflora,especially the Veillonia,were significantly reduced,but the conditional pathogens: Haemophilus-parainfluenzae,Porphyromonas-endodontalis,and spirochetes were significantly higher than those in the normal group.Gram-positive cocci and streptococcus and Gram-negative cocci and Velgobacteria were significantly reduced in the patient group.Gram-positive Bacillus and Haemophilus increased significantly,and the ratio of Bacillus or cocci in the oral microflora was changed.Gram-positive bacilli in Bacillus was increased,and gram-negative bacilli suchas Fusobacterium,Bacteroides,Prevotella,and Porphyromonas were significantly decreased,indicating that the proportion of negative and positive bacteria within the strain also changed.After treatment,the abundance of Streptococcus species in the genus was recovered,and the abundance of Haemophilus was reduced.The abundance ratio between the two genus also changed significantly,and the ratio of cocci or bacilli was recovered.The abundance and thermal map of species showed that the predominant species of RAU patients with latent fire in the spleen and stomach after treatment had a tendency to be close to that of the normal group,which was the most similar to the order level.AnaLysis of species complexity and diversityindicated that the species diversity of the patient group was higher than that of the normal group in Venn diagrams,and the species abundance was changed drastically.After treatment,the common operational taxonomic units(OTUs)in the normal group and treated patient group were increased.α diversity anaLysisshowed that the species complexity of the patient group was significantly greater than that of the normal group(P<0.05).The diversity in the treatment group showed a decreasing trend compared with the patient group,but the difference was not significant(P>0.05).β diversity anaLysisshowed that anaLysis of PCoA and NMDS in patient group was far from the normal group,while anaLysis of PCoA and NMDS of the treated group was close to that of normal group.The clustering tree data showed the significant difference and the longer clustering distancebetween the patient group and the normal group,the smaller difference between the treatment group and the normal group,and the shorter clustering distance was revealed.After the analysis of differences between groups in βdiversity index,it showed that the differences in size and discrepancies between the patient group and the normal group were significant,and syndromes were recovered after treatment,gradually sliding to the intermediate values,which indicated the therapeutic effect of Xie-huang-San.T-test analysis showed that the dominant species in the patient group and the normal group had significant differences in the levels of the phylum,genus,and species,especially in the species level differences.However,there were fewer species with significant differences between the treatment group and the normal group.The most significant differences between the two groups were-Haemophilus-parainfluenza(p=0.029)and Porphyromonas-endodontalis(p=0.046).The Porphyromonas in the treatment was very different from the patient group and the normal group(p=0.032 and p= 0.044).Functional gene analysisin the KEGG database showed that genetic expression of Ko00230(purine metabolism),Ko00520(amino acid and nucleoside glucose metabolism),Ko01230(amino acid biosynthesis),Ko02010(ABC translocator),Ko02024(organ-level quorum sensing),Ko03010(genetic information processing ribosomal translation),Ko04070(phosphatidylinositol signaling system),Ko04150(mTOR signaling pathway),Ko04151(PI3K-AKt signaling pathway),Ko04973(carbohydrate digestion and absorption)in the patient group were significantly lowcompared with those of the normal group.Among the upregulation,the expression of functional genes of Ko04973(carbohydrate digestion and absorption pathway)was significantly lower than that of the normal group.After treatment with Xie-huang-San,the expression of related metabolic function genes was up-regulated.In the database of functional abundance of eggNOG,the expression of functional homologous genes such as COG3039(transferase),COG3209(YD-repeat protein),and COG 3501(Rhs-Vgr protein)was significantly higher in the patient group than that in the normal group.The expression of functional homologous genes such as COG1132(ABCtransporter),COG0463(glycosyltransferase-2),EONG410ZMW6-NA and EONG410ZQAS-HTH-XRE was significantly lower than that in the normal group.The expression of EONG410ZMW6-NA and EONG410ZQAS-HTH-XRE was up-regulated after treatment with Xie-huang-San.In the CAZy function abundance database,the expression of functional genes such as GH2,GH20,GH38,GT10,GT51,CBM32,and CBM 48 in the patient group was significantly lower than that in the normal group;The expression of related metabolic genes such as GH38,GT51,GT10 and CBM48 in patients treated with Xie-huang-San was close to that of the normal group.Conclusion:(1)The fortifying spleen and purging fire method had a significant effect on relieving the clinical symptoms of RAU patients with latent fire in the spleen and stomach and reducing the long-term recurrence rate,no adverse reactions and side effects were observed.(2)The fortifying spleen and purging fire method can correct the oral immune dysfunctions,and has benign regulative effect on the structure and functional genes of oral microflora in RAU patients with latent fire in spleen and stomach,promote recuperative effect on abnormal gene expression and the establishment of a new oral meteorological balance.(3)The structure of oral microflora in RAU patients with latent fire in the spleenand stomach was significantly different from the normal individuals,The abundance of Haemophilus-parainfluenzae and Porphyromonas-endodontalis in RAU patients with latent fire in the spleen and stomach was significantly increased,which may be closely related to TCM latent fire in spleen and stomach.It is expected to be a characteristic strain of the syndrome. |
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